Cholinergic influences on intestinal propulsion were determined in vivo in fasted rats by measuring the movement of a nonabsorbable radioactive marker along the intestine following treatment with cholinergic drugs. The marker was instilled directly into the intestine via a previously implanted cannula. The direct effects of cholinergic drugs on intestinal contractions were determined in vitro using isolated segments of duodenum and jejunum. Neostigmine (0.1 mg/kg) produced a marked increase in intestinal transit that was blocked by atropine pretreatment (1.0 mg/kg) but not hexamethonium pretreatment (20 mg/kg). Atropine pretreatment alone significantly delayed transit while hexamethonium treatment alone did not affect intestinal transit. Neostigmine produced a concentration-dependent (0.3-30 microM) increase in contractions in both duodenal and jejunal segments in vitro. Prior incubation of the tissues with atropine (10(-7) M) blocked the neostigmine-induced contractions while prior incubation with hexamethonium (10(-6) M) did not. Contractions produced by substance P were not affected by atropine or hexamethonium. These data indicate that enhancement of cholinergic neurotransmission by neostigmine treatment increased intestinal propulsion and that this effect was mediated at muscarinic cholinergic receptors. Furthermore, inhibition of ongoing cholinergic transmission by atropine treatment reduced intestinal propulsion. The increase in transit produced by neostigmine may result from a stimulation of intestinal contractions. Cholinergic neurons are important mediators of intestinal propulsion in the rat as in other species.