The feasibility of utilizing fluorescence in situ hybridization with chromosome-specific DNA probes as the basis of an assay to detect aneuploidy and aneuploidy-inducing agents in interphase human lymphocytes has been investigated. The assay involves counting the number of hybridization regions in interphase cells to determine the number of copies of a specific chromosome of interest, 22,000 interphase nuclei from untreated 72-h lymphocyte cultures were examined following hybridization with probes for chromosomes 1, 7, 9, 17, X or Y. The combined frequencies of nuclei containing 0, 1, 2, 3 and 4 hybridization regions for the various autosomal chromosomes were 0.004, 0.084, 0.909, 0.003 and 0.001, respectively. Based on these frequencies, scoring 1000-2000 cells should allow detection of aneuploid cells with a 0.012 frequency of hyperdiploidy or a 0.11 frequency of hypodiploidy for a specific chromosome of interest (alpha = 0.05, beta = 0.80). This difference in test sensitivity is related to the higher frequency of cells with one apparent spot. A comparison of the ratio of hybridization region to nuclear area in the two-dimensional images used for this analysis indicates that an overlap of the two regions probably accounts for the high frequency of apparent monosomy observed in normal cells. Treatment with the aneuploidy-inducing chemicals, colchicine, vincristine sulfate and diethylstilbestrol resulted in significant dose-related increases in the number of nuclei containing 3 or more hybridization regions. Treatment with the clastogen sodium arsenite produced only a minor increase in apparently hyperdiploid cells whereas treatment with ionizing radiation, another potent clastogen, resulted in a significant increase in nuclei containing multiple hybridization regions. These results suggest that ionizing radiation is an aneuploidy-inducing agent under these conditions although chromosomal breakage within the hybridization region may account for a portion of the increased frequency of nuclei with multiple hybridization regions. These results indicate that the use of fluorescence in situ hybridization with DNA probes is capable of detecting aneuploid cells occurring at relatively low frequencies within a population of cells. Assays based on these techniques should facilitate a more rapid identification of aneuploidy-inducing environmental and therapeutic agents.