Human spermatozoa were incubated in albumin-containing Hepes-buffered modified Ringer's solution, in the presence or absence of externally supplied substrates. The acylated forms of carnitine were identified by bioautography. Incubation of the cells with propionate or n-valerate resulted in increased content of propionylcarnitine, but n-butyrate, isobutyrate, n-valerate, isovalerate, hexanoate or heptanoate did not result in the appearance of acetylcarnitine of chain length C4-C7. The addition of methionine, valine or isoleucine (whose catabolic pathways should produce propionyl-CoA) to the incubation medium did not increase propionylcarnitine. In all cases acetylcarnitine was the major acylcarnitine in human spermatozoa. The ratio of acetylcarnitine:carnitine remained relatively constant in spermatozoa incubated without external substrate for up to 4 h. No significant change in the ratio was observed when glucose, fructose or citrate were present in the incubation medium. Sorbitol decreased the ratio slightly and aspartic acid slightly increased it. A more pronounced increase in the ratio was caused by lactate or pyruvate. This increase was observed in motile spermatozoa but not in samples from asthenospermic men, indicating that metabolic utilization of pyruvate and lactate may differ in motile and immotile cells.