Testicular sperm biopsy combined with intracytoplasmic sperm injection (ICSI) allows men with azoospermia the possibility of fathering a child. However, little information exists on the risk of chromosome abnormalities in their sperm. Multicolor fluorescence in situ hybridization (FISH) analysis was used to determine the frequency of sperm diploidy and disomy for the sex chromosomes in six men with normal karyotypes and non-obstructive azoospermia. A new method using microwave decondensation and codenaturation of sperm nuclei yielded a much larger number of sperm nuclei for FISH analysis than our previous study of men with azoospermia. A total of 59916 sperm were analyzed; more than 9000 sperm were scored for each man. The men with nonobstructive azoospermia had an increased frequency of sperm chromosomal disomy for YY, XY, total sex chromosomal disomy, and diploidy compared with 18 normal controls, but only YY disomy reached statistical significance. One infertile man had a frequency of 3.8% XY disomy and 4.3% diploidy, which was 13-fold and 7-fold higher than control donors, respectively. Our results suggest that some men with nonobstructive azoospermia have a significantly increased frequency of sex chromosomal abnormalities than normal men, but that the overall frequency of abnormalities is similar to that found in infertile men with abnormal semen parameters.