Caffeine, pentoxifylline, 2-deoxyadenosine, cyclic adenosine monophosphate (cAMP), relaxin, adenosine, kallikrein, and calcium were compared for their ability to stimulate motility of cryopreserved sperm. Caffeine, pentoxifylline, and 2-deoxyadenosine significantly increased the percentage of motile sperm at 15, 30, 45, and 60 minutes after administration. Sperm velocity was significantly increased by caffeine at 0, 15, 30, and 45 minutes, and by pentoxifylline at 0, 45, and 60 minutes. Consistent stimulation was not observed for other chemicals. Caffeine, pentoxifylline, and 2-deoxyadenosine were then examined for their ability to provide motility stimulation after removal with washing. With the exception of caffeine, percent motility and velocity for stimulated and untreated sperm were similar after washing. A significant reduction in motility was observed at 48 hours after washing for caffeine. The percentage of hamster oocytes penetrated at 24 hours after washing was significantly reduced for caffeine, 2-deoxyadenosine, and pentoxifylline combined with 2-deoxyadenosine. Pentoxifylline-treated sperm showed no reduction in fertilizing capacity. These results indicate that, of the chemicals examined, pentoxifylline is superior for motility stimulation of cryopreserved sperm.