5-hydroxytryptamine (5-HT, serotonin) acts as a modulator of colonic motility and secretion. We characterized the action of the 5-HT precursor, 5-hydroxytryptophan (5-HTP), on colonic myenteric neurons and propulsive motor activity in conscious mice. Fos immunoreactivity (IR), used as a marker of neuronal activation, was monitored in longitudinal muscle/myenteric plexus whole mount preparations of distal colon 90 min after intraperitoneal (ip) injection of 5-HTP. Double staining of Fos-IR with peripheral choline acetyltransferase (pChAT)-IR or nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was performed. Injection of 5-HTP (0.5, 1, 5 or 10 mg/kg, ip) increased fecal pellet output and fluid content in a dose-related manner, with a peak response observed within the first 15 min post-injection. 5-HTP (0.5-10 mg/kg) increased dose-dependently Fos expression in myenteric neurons, with a maximal response of 9.9±1.0 cells/ganglion (P<0.05 vs vehicle-treated mice: 2.3±0.6 Fos-IR cells/ganglion). There was a positive correlation between Fos expression and fecal output. Of Fos-positive ganglionic cells 40±4% were also pChAT-positive and 21±5% NADPH-diaphorase positive in response to 5-HTP, respectively. 5-HTP-induced defecation and Fos expression were completely prevented by pre-treatment with the selective 5-HT₄ receptor antagonist, RS 39604. These results show that 5-HTP injected peripherally increases Fos expression in different populations of cholinergic and nitrergic myenteric neurons in the distal colon and stimulates propulsive colonic motor function through 5-HT₄ receptors in conscious mice. These findings suggest an important role of activation of colonic myenteric neurons in the 5-HT₄ receptor-mediated colonic propulsive motor response.