Fluorescence in situ hybridization using chromosome-specific DNA probes is rapidly becoming part of clinical laboratory practice for certain congenital and neoplastic disorders. Current legislation requires proficiency testing for clinical laboratory studies. To evaluate the efficacy of fluorescence in situ hybridization proficiency testing, we invited 19 representative institutions to participate in three pilot studies. One study used probes for the X and Y chromosomes to evaluate metaphase spreads and interphase nuclei. Another study used probes for bcr and abl to detect bcr/abl fusion in interphase nuclei in chronic myelogenous leukemia. The third study used a D22S75 probe to detect microdeletions in metaphase spreads from a patient with velocardiofacial syndrome. The results of these studies demonstrate that proficiency testing with fluorescence in situ hybridization is attainable using either metaphase or interphase preparations, and that either microscope slides or fixed cell pellets are suitable.