Isolated villus from human term placenta contains about 167 nmol of acetylcholine (ACh) per gram of wet tissue and releases about 35 pmol of ACh/g/min when it is suspended in Krebs-Ringer bicarbonate buffer at 37°C. Chronic doses of nicotine, or smoking, which modify ACh output from human placental villus, are known to retard fetal intrauterine growth. Since one of the functions of placental villi is nutrient transport, and since it has not been possible to obtain ACh-free villi, the effects of cholinergic blockade by high concentrations of ACh (2 × 10 −3 m), phospholine (5 × 10 −4 m), nicotine (2.5 × 10 −3 m), and atropine (10 −4 to 5 × 10 −4 m) on active uptake of α-aminoisobutyric acid (AIB) were studied to explain their antigrowth effects. High concentrations of ACh and nicotine decreased the rate of uptake of AIB by 34 and 41%, respectively. Atropine inhibited the AIB uptake by 29 and 61% at concentrations of 10 −4 and 5 × 10 −4 m, respectively. If all Ach were released from the syncytiotrophoblast, the concentration of ACh in the medium would be about 1.67 × 10 −5 m. At the highest concentration of atropine used, the concentration of the active antagonist, d(−) S-hyoscyamine, in the medium is 2.5 × 10 −4 m, which depressed AIB uptake by about 61%. Phospholine (an irreversible cholinesterase inhibitor) at 7 × 10 −6 m increased AIB uptake by 20%, but it decreased AIB uptake at higher concentrations, with or without exogenous ACh. Mecamylamine (10 −4 m) and d-tubocurarine (10 −4 m) did not influence the AIB uptake. Among the pharmacological agents studied, nicotine increased the release of endogenous placental ACh, while atropine decreased ACh release. These observations indicate that endogenously released ACh exhibits a muscarinic effect on the placental villus and facilitates the uptake of amino acids. Blockade of the facilitating effects of ACh on amino acid uptake by placenta for long periods during pregnancy may result in a retardation of fetal growth.