The present study was undertaken to evaluate the 24-hour periodicity in serum prolactin levels subsequent to transecting the ascending serotonergic system or ablating the suprachiasmatic nuclei (SCN) of adult (185 g) female rats. The ascending serotonergic system was transected at the level of the rostral mesencephalon using a 1.5 mm wide knife and the SCN was ablated with a modified Halász-Pupp knife. The effect of raphe transection (RT) or SCN ablation on the circadian rhythm in nonstress serum prolactin levels was assessed 45 days after surgery by measuring with radioimmunoassay the serum prolactin concentration in serial blood samples obtained from the tail vein of lightly restrained rats. Serum prolactin concentration in control and RT animals showed circadian periodicity; peak levels occurred during the midafternoon, 4 h prior to the dark phase of the lighting regime (lights on at 4 a.m., off at 6 p.m.). The amplitude of the fluctuations of both groups varied markedly and were related to the estrous cycle. However, RT animals showed a reduced amplitude in the rhythm. In controls peak prolactin levels on days of proestrus and estrus were 15–20 times higher than on days of diestrus. In contrast, serum prolactin levels in SCN-ablated rats did not vary with a circadian periodicity but rather showed random, low amplitude fluctuations. Reproductive cyclicity was also abolished by SCN ablation, i.e., SCN-ablated rats presented persistent vaginal cornification. These data indicate that circadian periodicity in serum prolactin levels is not compromised by sectioning the ascending serotonergic fibers but is abolished after ablating the S