The main objectives of this investigation are (1) to demonstrate the existence of a functional cholinergic system in spermatozoa; (2) to study the role of acetylcholine (ACh) in sperm motility, viability and fertilizing capacity; and (3) to develop inhibitors of sperm choline acetyltransferase (ChA) and sperm maturation. Our studies have indicated that ACh system is present in human, bull, rat, and mouse spermatozoa. According to our studies, ChA is located in a bound state on the outside surface of the spermatozoa. Therefore, ACh seems to be synthesized on the surface of sperm tail and acts locally to induce motility. There is a positive relationship between the inhibition of human sperm motility and the inhibition of ChA inhibitors in vitro. Bull spermatozoa which did not exhibit motility contain very low levels of ACh and ChA. Spermatozoa contain both ATP citrate lyase and acetate thiokinase which form acetylcoenzyme A from citrate and acetate respectively. On a quantitative basis, ATP citrate lyase seems to be the most important enzyme for the formation of ACoA, which is utilized for the formation of ACh. Our investigations which are in progress include: (1) effects of tertiary ChA inhibitors on sperm motility; (2) distribution of ATP citrate lyase and acetate thiokinase in sperm fractions; (3) binding of cholinergic agonists and antagonists to sperm fractions; (4) sources of choline which is utilized in ACh synthesis; (5) Antifertility effects of nicotine and ChA inhibitors in rats; and (6) effects of antifertility agents (cyproterone acetate, alpha-chlorhydrin and trimethylphosphate) on the levels of ACh, ChA, acetate thiokinase and ATP citrate lyase in epididymal spermatozoa in the rat. The results of this investigation may be useful to (1) explain the role of ACh in the regulation of sperm motility, (2) understand certain types of infertility in men who ejaculate immotile sperm, (3) explain transient infertility among male smokers, and (4) develop antifertility agents.