Intensely labeled nerve cell bodies were identified by radioautography within the pars ventralis of nucleus dorsomedialis hypothalami (hdv), following intraventricular perfusion with 10(-5) or 10(-4) M tritiated serotonin [3H]5-HT in adult rats pretreated with a monoamine oxidase inhibitor. This selective reaction, which involved approximately 1000 neurons on each side of the third ventricle, was unaltered by concomitant administration of 10(-3) M non-radioactive norepinephrine, and was absent after intraventricular injection of 10(-5) or 10(-4) M tritiated norepinephrine. The 3H-labeled 5-HT nerve cell bodies were loosely grouped within the inner and caudal half of the hdv, and appeared morphologically similar to the unreactive neurons among which they were interspersed. Within the same region, numerous labeled axonal varicosities were also detected, which were never found in synaptic contact with the reactive cells. If the 3H-labeled 5-HT neurons contain endogenous 5-HT, they might constitute an intrinsic source of 5-HT innervation in the adult rat hypothalamus.