The frequencies of aneuploid and diploid sperm were determined in a 47,XYY male using multi-color fluorescence in situ hybridization (FISH) analysis, and compared with those from 10 control donors. A total of 30,078 sperm from the patient was scored, 15,044 by two-color FISH for chromosomes 13 and 21, and 15,034 by three-color FISH for the sex chromosomes using chromosome 1 as an internal autosomal control for diploidy and lack of hybridization. The frequencies of X-bearing (49.73%) and Ybearing sperm (49.46%) in control males were not significantly different from the expected 50% ( 2 -test for goodness of fit). The ratio of 24,X (50.60%) to 24,Y sperm (48.35%) in the patient, however, was significantly different from the controls (P = 0.0144, 2test for independence) and from the expected 1:1 ratio (P = 0.0055, 2 -test for goodness of fit). There was no significant increase in the frequency of diploid sperm when compared with the controls ( 2 -test for independence). Significantly increased frequencies were found for 24,YY (0.07% vs. 0.02%, P = 0.0009) and 24,XY (0.44% vs. 0.29%, P = 0.0025), but not for 24,XX (0.05% vs. 0.05%, P > 0.05), 24,+13 (0.07% vs. 0.07%, P > 0.05) or 24,+21 sperm (0.21% vs. 0.18%, P > 0.05) in the 47,XYY male when compared with control donors ( 2 -test for independence). Our results support the theory that loss of the extra Y chromosome occurs during spermatogenesis in most cells. In this XYY patient there was a significant increase in the frequency of sperm with sex chromosomal ab-normalities but no suggestion of an interchromosomal effect on autosomes. All 3-color FISH studies in the literature demonstrate a significantly increased risk of gonosomal aneuploidy in XYY males, with the risk being on the order of 1%. Am. J. Med. Genet. 93:40-46, 2000.