A subpopulation of nerve fibers in the rat pituitary intermediate lobe (IL) have been shown to exhibit colocalization of serotonin (5-HT-IR) and tyrosine hydroxylase immunoreactivities and they are sensitive to neurotoxins specific to catecholamine neurons. This study was set out to examine the uptake and synthesis mechanisms of serotonin in these fibers. We developed an in vitro technique in which the neurointermediate lobe explants were incubated (14 and 48 h) in the presence of various drugs and serotonin was subsequently visualized by immunohistochemistry. Control incubation in the presence of serotonin (10(-6) M) resulted in a rich plexus of 5-HT-IR fibers in both posterior and intermediate lobes. Fluoxetine and citalopram (10(-6) M and 10(-5) M), inhibitors of 5-HT transporter, did not affect 5-HT-IR in the IL fibers, unless they were used in concentrations high enough (10(-4) M and 10(-3) M) to block unspecifically a number of monoamine transporters. The same applied for desipramine (10(-5)-10(-7) M), an inhibitor of the noradrenaline transporter. However, cocaine (10(-5)-10(-6) M) blocked serotonin uptake into these terminals, suggesting that serotonin uptake occurs through a dopamine transporter. Incubation of the IL in presence of L-tryptophan (10(-4) M) did not result in 5-HT-IR in the IL fibers showing colocalization of 5-HT-IR and tyrosine hydroxylase, which suggests that these fibers do not synthesize serotonin. The present results suggest that serotonin is taken up into the IL terminals by a dopamine transporter and is not synthesized in them, at least in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)