The presence of functional muscarinic-cholinergic receptors on at least some T lymphocytes is suggested by the existence of saturable, high-affinity, specific muscarinic binding in T cell-enriched, but not in B cell-enriched, cell suspensions and by observed cholinergic effects on lymphocytes, (e.g., increased lytic capacity of cytotoxic lymphocytes preincubated with muscarinic agents). In this study, we used flow cytometry and a fluorescent probe of membrane potential, the cyanine dye 3,3'-dihexyloxacarbocyanine iodide, to examine the effects of cholinergic agonists and antagonists on the membrane potentials of lymphocytes in T cell-enriched and B cell-enriched suspensions. Acetylcholine (AcCho) and carbamoylcholine (CbmCho) depolarized the membranes of T cells, but not of B cells; the maximal depolarization was produced by 10 nM AcCho or by 1 nM CbmCho. Depolarization following exposure to these concentrations of agonists was maximal by 5-8 min; T cell membrane potentials returned to control values by 13-15 min. Less marked depolarization was produced by 100 nM AcCho and 10 nM CbmCho; 100 pM CbmCho was only slightly less effective than 1 nM CbmCho, and the depolarization persisted 12 min after exposure. Depolarization induced by 1 nM AcCho was abolished when AcCho was combined with 10 nM atropine but not when AcCho was combined with 1 nM atropine or 200 nM d-tubocurarine. The time course of the membrane potential response and its dependence on the relative concentrations of AcCho and specific cholinergic blocking agents correlate well with both binding studies and biological effects. Our results provide evidence that T lymphocytes have functional muscarinic receptors; the flow cytometric method should be generally applicable to studies of the electrophysiology and pharmacology of receptor-ligand interactions.