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Flow cytometric analysis of enzymatically decondensed, DAPI-stained spermatozoa was performed to confirm the suspected production of unbalanced spermatozoa in heterozygous rams carrying a 1;20 translocation. High-precision flow cytometry (coefficient of variation, 0.6-0.8%) with a PAS II flow cytometer depicted Y- and X-chromosome-bearing spermatozoa from three cytogenetically normal rams as two distinct peaks. The difference in DNA fluorescence intensity between the gonosomes averaged 4.8%. Analysis of sperm samples from three heterozygous 1;20 translocation carriers yielded histograms with five peak distributions. The individual peaks were attributed to spermatozoa with a normal, balanced, and unbalanced chromosomal status. Peaks within Y- and X-spermatozoa populations were distributed in a ratio of 1:2:1 and were almost completely separated, with a coefficient of variation of 0.5-0.6%. Owing to the relative size of the translocated chromosomal segment (2.4% of the total DNA content, as determined from the flow cytometric data), histograms with five instead of the expected six peaks were observed.