Nitric oxide (NO) synthase activity in rat isolated longitudinal muscle myenteric plexus preparations of the small intestine was determine by measuring the accumulation of 3H-L-citrulline during 30 min incubation with 3H-L-arginine. In untreated preparations a significant amount of 3H-L-citrulline accumulated in the tissue, about 2000 dpm/30 mg per 30 min, accounting for about 1.7% of the tissue radioactivity. Intermittent electrical field stimulation (15 Hz, 10 s trains with 10 s intervals for total of 20 min) caused a threefold increase in 3H-L-citrulline accumulation. The NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) reduced the spontaneous accumulation of 3H-L-citrulline by 65% and prevented the electrically evoked increase. Removal of extracellular calcium or addition of tetrodotoxin blocked the electrically evoked increase in 3H-L-citrulline accumulation without affecting spontaneous accumulation. Application of the calcium ionophore A 23187 (10 mumol/l) or 45 mmol/l) or 45 mmol/l potassium caused a twofold increase in the accumulation of 3H-L-citrulline. The muscarine receptor agonist oxotremorine (1 mumol/l) had no effect on spontaneous accumulation of 3H-L-citrulline, but inhibited the electrically evoked increase by about 50%, and this effect was blocked by scopolamine. A substantial amount of 3H-L-citrulline (15000 dpm) accumulated also in the incubation media, and this was increased 1.7-fold by the presence of A 23187 and 2.7-fold by electrical stimulation. However, electrically evoked increase in 3H-L-citrulline was not prevented by tetrodotoxin, in contrast to observation on tissue levels. In conclusion, during incubation with 3H-L-arginine tissue levels of 3H-L-citrulline in rat isolated longitudinal muscle myenteric plexus preparations, but not accumulation in incubation media may be used as a biochemical marker of the activity of nitrergic intestinal neurons which appear to be inhibited via muscarine receptors.