Progesterone stimulates prolactin secretion in estrogen-primed women and monkeys. We hypothesize that this effect is neurally mediated since pituitary lactotropes do not contain progestin receptors (PR). In rodents, progesterone enhances hypothalamic serotonin (5HT) content, and both progesterone and 5HT stimulate prolactin and LH secretion. However, it was not known whether progesterone acts directly on 5HT neurons or through other neurons. Using a double immunocytochemical procedure, we show that 5HT neurons in macaque contain PR and thus are a progestin target system. Midbrain tissue blocks were obtained from two female monkeys and immersion-fixed prior to freezing and sectioning. PR was detected with a monoclonal antibody against human PR (B39) bridged to horseradish peroxidase and developed in diaminobenzidine. PR immunoreactivity appeared as a brown reaction product which localized in the nuclei of individual neurons. 5HT was detected with an antiserum generated against a conjugate of 5HT and BSA bridged to alkaline phosphatase. 5HT immunoreactivity appeared as a blue reaction product in the cytoplasm and axons of the pontine raphe nucleus. Neurons containing both nuclear reaction product for PR and cytoplasmic reaction product for 5HT were observed in both the dorsal and ventral aspects of the midbrain raphe nucleus as well as the raphe magnus. In summary, progesterone can have a direct action on 5HT neuronal function and thereby influence those endocrine and affective systems under serotonergic control.