In the male reproductive tract, very high concentrations (mmol l−1) of free l-carnitine and acetyl-l-carnitine are found in the epididymides, seminal plasma and spermatozoa. It has been reported that the uptake of free l-carnitine by spermatozoa might be related to the epididymal maturation of the sperm membrane, since a greater uptake was found by caput than by cauda spermatozoa in vitro. However, the free l-carnitine concentrations estimated inside the gametes were never greater than those of the surrounding medium. In this study, we investigated the mechanism of transport of free l-carnitine and its ester acetyl-l-carnitine, through the plasma membrane of mature and immature epididymal boar spermatozoa. In vitro, we found a passive diffusion of both compounds to the spermatozoa, whatever the maturation stage. The spermatozoa might progress in the epididymal lumen and accumulate high amounts of free l-carnitine. The active uptake of free l-carnitine occurs only across epididymal mucosa. These results are in agreement with those reported on cells of other organs that exchange pharmacological free l-carnitine concentrations (mmol l−1) by a passive mechanism through the plasma membrane. The acetylation of high amounts of free l-carnitine inside the spermatozoa was found only in caudal spermatozoa. This result suggests that oxidative metabolism (producing acetyl CoA) might be more active in mature cells. The acetyl-l-carnitine added to the incubation medium of boar spermatozoa was hydrolysed. Enzymatic activity of the sperm membrane is low and this may partially explain the low concentrations of acetyl-l-carnitine found in the caudal epididymal plasma.