A method for the identification of Mycobacterium species using reversed-phase high performance liquid chromatography (RP-HPLC) was developed. The fingerprints library was constructed on the basis of RP-HPLC chromatograms of the mycolic acids derivatives from 49 Mycobacterium species cultures. Two inoculation loops of Mycobacterium, for fast growth with one week incubation or for slow growth with three weeks incubation, were saponified for 1 h and stocked at 4 degrees C. The mycolic acids from each culture of Mycobacterium species were acidified, extracted, derivatized, and analyzed by the RP-HPLC method. On the basis of the HPLC patterns of relative retention time and relative peak height, the identifications of Mycobacterium species were performed. This established method has a good precision of retention times with the relative standard deviations (RSDs) ranging from 0.13% to 1.07%. The mycolic acids fingerprints library of HPLC patterns was set up, including 49 species that were recorded in "Bergey's Manual of Systematic Bacteriology". Three patterns were observed from the chromatographic behaviors of mycolic acids derivatives, including single peak-cluster, double peak-clusters, triple and multiple peak-clusters. Forty-one species were successfully identified according to the different relative retention times of the peaks and the relative peak heights. The established method can identify Mycobacterium species with rapidity and high reliability.