Single color fluorescence in situ hybridization (FISH) has been utilized on sperm to estimate nondisjunction rates for chromosomes 1, 12, 15, 16, X and Y. Using single-color FISH, one cannot distinguish nonhybridization from nullisomy nor disomy from diploidy. In order to provide an internal control, a multicolor FISH strategy was employed. Satellite probes specific for 13 human chromosomes were used on multiple semen samples from two normal donors. Two or three probes were hybridized simultaneously and scored by two independent observers. Over all experiments, 40,641 sperm were analyzed. The majority of autosomes had no significant difference in aneuploidy between chromosomes or between donors. However, a significant difference was observed for chromosome 18 between donors (χ22 = 7.078, 0.025 < P < 0.05). Additionally, no significant difference was found between donors for sex chromosome aneuploidy. The frequency of sex chromosome aneuploidy was similar to that seen in paternally derived 47, XXY and 47, XYY conceptuses. Furthermore, 0.15% of sperm were found to be diploid. Based on the results of this study, as much as 19% of all sperm may be chromosomally abnormal. This method proved to be useful for determining aneuploidy of human chromosomes in sperm and valuable in exploring whether individual differences of nondisjunction exist