L-Carnitine and acetylcarnitine concentrations were determined in spermatozoa and seminal plasma from 15 men, in both fresh ejaculate and frozen-thawed semen with cryoprotective medium. Sperm motility was also evaluated. In fresh samples, the levels of carnitine and acetylcarnitine in seminal plasma were comparable whereas in spermatozoa, acetylcarnitine predominated. Cryopreservation did not change the carnitine and acetylcarnitine levels in seminal plasma nor the carnitine concentration in spermatozoa; by contrast, the acetylcarnitine level in spermatozoa was decreased in 14 cases (110 +/- 8 versus 210 +/- 20 nmol/10(8) cells). This decrease in acetylcarnitine content was greater during semen dilution in cryoprotectant than after the freezing/thawing process. Motility was also decreased in all cases after the freezing/thawing process. These results suggest that acetylcarnitine recovery in spermatozoa is further evidence of the deleterious effect of the cryoprotective medium in the cryopreservation of semen.