The effects of 5‐hydroxytryptamine (5‐HT) on the longitudinal smooth muscle from the rumen and reticulum of the bovine forestomach were investigated. 5‐HT (0.25–490 μM) caused a contraction and a relaxation of the ruminal strips while it produced only an excitatory effect on the reticular strips. These effects were not affected by tetrodotoxin, hexamethonium, atropine or morphine, but were blocked by methysergide, LSD‐25 or phenoxybenzamine. 5‐HT potentiated the contraction evoked by stimulation of the intramural cholinergic nerves but did not show any effect on the relaxation produced by the non‐adrenergic inhibitory nerves' excitation. The 5‐HT‐induced potentiation was not affected by morphine, LSD‐25, methysergide and hexamethonium or high concentration of nicotine. Nicotine and dimethylphenylpiperazinium also caused a transient augmentation of the nerve‐mediated contraction, but these effects were abolished by the competitive ganglionic blockers. The evoked contraction was depressed in high‐Mg2+solution, but this depression was antagonized partly by 5‐HT. The affinity of the cholinomimetics to post‐synaptic muscarinic receptor was not affected by 5‐HT. It is concluded that contractions or relaxations of bovine forestomach strips induced by 5‐HT are mediated through activation of D‐receptors in the smooth muscle, and the 5‐HT‐induced potentiation of the evoked contraction may be elicited through presynaptic neural eff