The epididymal compounds taurine, hypotaurine and inositol, and the antioxidants carnosine and ascorbic acid, were added to Tris-based diluents containing varying concentrations of glycerol, and their effect on the post-thaw motility characteristics and fertility of ram spermatozoa was examined. Overall, the post-thaw motility characteristics of spermatozoa were better when semen was frozen in the presence rather than in the absence of glycerol. Only taurine protected spermatozoa during cryopreservation; the presence of 25 mM or 50 mM taurine significantly improved the post-thaw percentage of motile spermatozoa but this had no effect on fertility after cervical or laparoscopic insemination of ewes. Increasing the concentration of taurine to more than 100 mM significantly reduced the percentage of motile spermatozoa, compared with the lower concentrations of the amino acid. The presence of more than 50 mM carnosine or ascorbic acid significantly reduced all motility characteristics compared with the control diluent. Given that hypotaurine, carnosine, or ascorbic acid did not improve post-thaw motility, the cryoprotective effect of taurine may be attributable to its osmoregulation rather than to its antioxidant properties.