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This study evaluated the percentage of Y-chromosome bearing spermatozoal (%Y-CBS) variation from ejaculates within individual males using two experiments. In the first experiment, six ejaculates were taken from each of five sexually rested (>30 days) Holstein bulls. Ejaculates were processed separately and stored in liquid nitrogen. From each ejaculate, five straws were thawed and equal sperm number, pooled samples were constructed using hemacytometer counts. Individual ejaculate DNA samples were extracted and quantified by spectroscopy. AY-chromosome specific segment was amplified by polymerase chain reaction (PCR) and the product separated by gel electrophoresis. Ethidium bromide stained bands were detected by image analysis and equated to a 50 %Y-CBS pool. In the second experiment (91 days), two ejaculates were collected from sexually rested (>21 days) bulls weekly and two ejaculates were collected from bulls every 21 days. Specific Y-chromosome (SRY) and X-chromosome (factor IX, F9) sperm DNA was amplified by PCR and the products separated by gel electrophoresis. Ethidium bromide stained bands were detected by image analysis and compared to a standard curve constructed from pure SRY and F9 PCR product. The log ratio (SRY/F9) of the corrected intensity densities were used to estimate the percent Y-chromosome DNA bearing spermatozoa (%Y-CDBS) in each ejaculate using inverse regression procedures. In Experiment 1, ejaculate differences for the first collection ranged from 17 to 71 %Y-CBS. Differences remained large for the second ejaculates and lessened for the third and fourth collections. Differences were least for the last two collections. Sperm head area also fluctuated. In Experiment 2, collection frequency affected the pattern of %Y-CDBS response. In bulls collected weekly, %Y-CDBS changed in a sinusoid fashion with a period of about 13.5 days. For bulls collected on a 21-day interval, %Y-CDBS ejaculate differences were high in the first ejaculate after sexual rest. Maximization of %Y-CDBS variation between ejaculate and its identification by PCR would allow ejaculate selection to be used to the alter the sex ratio in producers' calf crops.