The secretory profile of prolactin and oxytocin in response to suckling stimuli by litters was studied in unanesthetized and urethane-anesthetized lactating rats. Serum prolactin levels were determined by radioimmunoassay. Oxytocin released at milk-ejection reflex was monitored by the changes in the intramammary pressure and/or the characteristic pup's reaction associated with the milk ejection. Serum prolactin concentrations began to rise earlier than the first milk ejection in unanesthetized rats, but they were never elevated without the appearance of milk ejections in urethane-anesthetized rats. Pulsatile fluctuation in serum prolactin levels at 6-15 min intervals was observed in the nursing period when 10 pups were suckling continually. The intermittent milk-ejection reflex occurred not always but preponderantly (64-91%) when the serum prolactin levels were at the nadir of the fluctuation. Injection of an estimated dose of oxytocin released at each milk ejection (1 mU) did not change the serum prolactin levels. These results indicate that the mechanism for prolactin release may be more susceptible to the effects of anesthesia than that for oxytocin release in response to the suckling stimuli and that the release of both the hormones is pulsatile in nature and be influenced by a common biological clock during the nursing period.