The heart rate of the isolated, perfused, working rat heart was significantly and equally depressed by 1 X 10(-6)M acetylcholine (ACh) and by 6 X 10(-5)M 4-ketoamyltrimethylammonium (4K), a cholinomimetic agonist. Dimethyl sulfoxide (DMSO) (10 microliter/ml, 140 mM) strongly potentiated the effect of ACh but did not alter the effect of 4K. DMSO (10 microliter/ml, 140 mM) strongly potentiated the effect of ACh but did not alter the effect of 4K. DMSO (10 microliter/ml, 140 mM final concentration) alone had no significant effect upon heart rate when added to the perfusate in incremental additions of 1 microliter X (ml perfusate)-1 X min-1 over a 10-min period. The specific activity of atrial homogenate cholinesterase was 48.8 +/- 3.46 nmoles X min-1 X (mg protein)-1 (mean +/- S.E.M.), 38.2 +/- 1.60 for butyrylcholinesterase, and 11.2 +/- 0.86 for acetylcholinesterase (AChE). True AChE activity (measured in the presence of a maximally effective concentration of tetraisopropylpyrophosphoramide) had a Vmax of 13.4 +/- 0.17 nmoles X min-1 X mg protein)-1 and an apparent Km value of 1 X 10(-4)M acetylthiocholine. At this Km substrate concentration, DMSO inhibited atrial AChE activity (I50 = 9 microliter/ml). At the concentration tested, DMSO inhibited atrial AChE and potentiated ACh effects.