Bartonella species are pathogens of emerging and reemerging significance, causing a wide array of clinical syndromes. In North America and Europe, they are increasingly recognized as a cause of culture negative endocarditis, neuroretinitis, and disease among homeless, HIV-infected, and other immunosuppressed individuals. In South America, bartonellosis continues to plague those in endemic regions and poses a significant threat to travelers in these areas. As the clinician is increasingly faced with these illnesses, which may be difficult to diagnose, laboratory techniques to confirm or refute the diagnosis are becoming increasingly important. Culture methods have improved over the past decade demonstrating increased sensitivity, but still require prolonged periods before isolation of the organism. Specimen handling, media selection, and growth conditions all may affect results and must be optimized in order to provide the highest likelihood of recovering the organism. Pure culture of the bacteria not only provides morphologic information, but also provides material for further diagnostic testing. Work with liquid media, which may provide a more rapid means of cultivation has shown some promise and should continue to be pursued. Improved blood culture techniques were a primary factor in the discovery of Bartonella endocarditis and continued improvements will likely demonstrate further clinical insights. Serologic testing for B henselae infections has become the cornerstone of clinical diagnosis, replacing the skin test that was poorly standardized and posed a potential risk to the patient. Immunofluorescence assays have been well characterized and validated in clinical trials, however they are not universally available. Vero cell cocultivated antigens appear to provide higher sensitivity and specificity when compared with agar-derived antigens. IFA assays are inherently difficult to perform, requiring significant expertise to provide reproducible results. On the contrary, enzyme immunoassays offer ease of use and a high level of reproducibility, however ideal antigens for use in the diagnosis of Bartonella infections have not been clearly identified. Continued work to define antigenic targets of the human response to infection and incorporation of these into a widely available EIA will provide a cost-effective tool for the clinician and epidemiologist alike. Due to the close phylogenetic relationship of B henselae and B quintana, differentiation between these species by serologic means may prove difficult. Molecular techniques including PCR offer high sensitivity and specificity, rapid availability of information, and the ability to differentiate Bartonella organisms at the highest level. Results of studies to date are promising and as methods are refined it will be important to conduct clinical studies to define the role of these assays. In disseminated Bartonella infections such as bacillary angiomatosis, peliosis, endocarditis, and urban trench fever, PCR currently offers the ability to establish the diagnosis when other tests may be unrevealing. For CSD, this technique should be used as a confirmatory technique when the diagnosis is unclear by other means. PCR analysis of blood specimens offers a minimally invasive approach to diagnosis, but clinical data are scarce and further studies are needed. As DNA microarrays move into the clinical arena, specific hybridization probes may allow improved identification and differentiation of Bartonellae at the molecular level.