The role of endogenous prolactin (PRL) in the control of testosterone (T) secretion and T responses to LH treatment was evaluated in adult male rats. Rats were actively immunized three times against ovine PRL in Freund's adjuvant-saline mixture (PRL-IMM rats), and control rats were treated with adjuvant-saline mixture (ADJ-CON rats). On day 110 after initial immunization, rats in each of these two groups were divided into three subgroups. Rats in subgroups 1 and 2 were injected with saline while those in subgroup 3 received 200 micrograms ovine PRL in saline, twice a day for a total of 7 injections. On day 113, the seventh injection was given 3 h before the termination of the experiment. On the same day, 2.5 h before the rats were sacrificed, rats in subgroups 1 and 3 were treated with saline; animals in subgroup 2 received 25 micrograms ovine LH in saline. Blood samples were obtained throughout the study, and sera were used for measurement of PRL antibodies, gonadotropins, progesterone (P), and T. PRL antibodies were detected in the sera of all rats actively immunized with PRL. Administration of PRL increased serum T levels in ADJ-CON rats, and this effect was eliminated in rats actively immunized against PRL. LH treatment significantly increased serum T levels in ADJ-CON rats. In PRL-IMM rats, this increase was attenuated while circulating P concentrations were elevated. These data demonstrate that PRL treatment can increase T secretion and that endogenous PRL is required for the complete expression of the stimulatory action of LH on T secretion in adult male rats.