Relaxin and prostaglandin E2 (PGE2) are present in human semen and have been shown to affect sperm motility. The authors further examined the effects of porcine relaxin and PGE2 on the motility of human spermatozoa. A dose-response study revealed that PGE2 at a concentration of 25 μg/ml is most effective in improving the motility of washed human sperm. Relaxin (100 ng/ml), PGE2 (25 μg/ml), or the two combined have no effect on the motility of spermatozoa in fresh, normal semen, suggesting that the constituents of fresh semen are optimal for motility. Relaxin and PGE2 individually improve the motility of washed spermatozoa. However, relaxin, but not PGE2, improves the motility of sperm in semen incubated at 37°C for 5 hours (aged). In contrast to the individual substances, a combination of relaxin + PGE2 has no effect on the motility of washed spermatozoa or aged spermatozoa, suggesting that these two substances antagonize each other's action on sperm motility. The presence of both relaxin and PGE2 in seminal plasma with normal motility spermatozoa suggests that other factors in seminal plasma regulate the effects of these substances on sperm motility.