For successful fertilization, a functionally constituted sperm plasma membrane is necessary, and this is clearly dependent on the sperm maturation process. The latter involves a series of complex changes which result from a sequence of events occurring at different points within the epididymis. The transit time through the epididymis can be influenced by external factors such as sexual stimulus and ejaculatory frequency. The present work was undertaken to determine changes in ram sperm viability and other sperm quality characteristics in relation to ejaculatory frequency. Three successive ejaculates were collected from rams during three different abstinence periods (collected every day, every 2 days and every 3 days). Cell viability (membrane integrity determined by fluorescence staining), progressive individual motility, and other in vitro parameters of sperm quality were evaluated. Second ejaculates showed the highest cell viability of the three periods studied, and increased as the abstinence period lengthened. The maximum proportion of viable cells (average 60%) was obtained in the second ejaculate after an abstinence period of 3 days. Likewise, overall and progressive individual motilities were higher in second ejaculates, the maximum value being 70% after 3 days of abstinence. The percentage of damaged or acrosome-reacted spermatozoa was greater after 1 day of abstinence than after the other periods analysed, whereas the first ejaculate showed the highest value in all periods. Differences in ejaculate volume were correlated strongly with both variables considered (abstinence period and ejaculate number). In the third ejaculate, about 27% more volume was obtained after 3 days of abstinence than after abstinence for 1 or 2 days. Sperm concentration increased significantly as the abstinence period lengthened, and also decreased significantly with ejaculate number in all cases. Therefore, the total number of spermatozoa in the ejaculate was clearly dependent on the abstinence period and the ejaculate number. In conclusion, the results obtained suggest that using the second and/or a mixture of second and third ejaculates would improve the results in artificial insemination and in fertility studies. In addition, the use of better quality semen would facilitate progress in semen cryopreservation studies.