These investigations were undertaken to show the presence of acetylcholine (ACh) and other quaternary ammonium compounds and choline acetyltransferase (ChA) activity in mammalian spermatozoa. The spermatozoa from the fresh ejaculates of the bull and man were washed with calcium-free Krebs-Ringer phosphate solution and extracted with acetonitrile. The quarternary ammonium compounds from the extracts were subjected to pyrolysis gas chromatography. In the gas chromatogram of the quaternary ammonium compounds of bull spermatozoa, two peaks were found which represented 2-dimethylaminoethyl acetate and 2-dimethylaminoethyl propionate. In the gas chromatogram of the quaternary ammonium compounds of the human spermatozoa, a peak for 2-dimethylaminoethyl acetate was found. These observations suggest that ACh and propionylcholine (PCh) occur in bull spermatozoa and that ACh occurs in human spermatozoa. Bull spermatozoa contained 4.27 ± 1.41 pmoles (mean ± S.E.M.) ACh/10 6 cells and 1.47 ± 0.48 pmoles PCh/10 6 cells, and human spermatozoa contained 28.57 ± 3.38 pmoles ACh/10 6 cells. ChA activity was determined in the washed spermatozoa by a radiometric assay. Incubation of the homogenates of the sperm cells with [ 14C]acetylcoenzyme A and choline resulted in the synthesis of [ 14C]ACh, which was separated, using a column of an anion exchange resin, and assayed. ChA activity in bull spermatozoa was 200.19 ± 32.08 pmoles ACh synthesized/10 6 cells/10 min, and ChA activity in human spermatozoa was 130.90 ± 14.19 pmoles ACh synthesized/10 6 cells/10 min. The specific activity of ChA in the tails of bull spermatozoa was about five times higher than in the heads or midpieces.