Dually perfused human placental lobules were employed to study the effects of vesamicol (an inhibitor of acetylcholine (ACh) storage) and physostigmine (an inhibitor of cholinesterase breakdown of ACh) on ACh output from fetal vessels and on materno-fetal transfer of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB). Bilateral perfusion with vesamicol (100 mumol L-1) or omission of physostigmine (2 x 7 mumol L-1) from the perfusate significantly reduced the output of ACh. However, neither drug had any significant effect on AIB transfer. Thus, the results of this study do not provide evidence for a close relationship between the placental cholinergic system and materno-fetal AIB transfer.