The aim of this study was to test the suitability of "pattern analysis" for the exploration of data provided by computer-assisted semen analysis methods. Data sets derived from the examination of boar sperm responses to bicarbonate and caffeine (measurements on 3208 spermatozoa) and from studies of semen cryopreservation in Mohor gazelles (7278 spermatozoa) were reanalyzed. A nonhierarchical classification method was used to generate initial subgroups of spermatozoa (9 for boar, 13 for gazelle). The subgroup centroids were fused, yielding three boar sperm subpopulations and four gazelle sperm subpopulations distinguished by sperm behaviors. Bicarbonate and caffeine both induced major transitions (p < 0.0001) of boar sperm behavior, detected as shifts in group membership (from group 2, i.e., active but nonlinear movement, into group 1, i.e., linear, rapid movement). Some spermatozoa (approximately 3%) were refractory to both caffeine and bicarbonate. The gazelle sperm subpopulation structure was affected by the inclusion of equex (sodium triethanolamine lauryl sulfate) in the cryoprotective diluents. Equex suppressed the appearance of spermatozoa with erratic behavior (p < 0.0001; high curvilinear velocity, low linearity, low straight-line velocity) after cryopreservation. The proportion of these erratic spermatozoa was positively correlated with animal age (r = 0.68, p = 0.029). Pattern analysis revealed novel aspects of the data not seen in the original investigations and usefully supplemented the more standard data analysis approaches.