Prolactin (PRL) secretion in the female rat is regulated by an endogenous stimulatory rhythm (ESR), which is normally under the tonic inhibition of dopamine (DA). The ESR consists of a nocturnal (N) component which peaks at approximately 03.00 h, and a diurnal (D) component which peaks at approximately 17.00 h. This ESR has been shown to be present in ovariectomized and cervically stimulated rats. We have proposed that the ESR is continuously present in the female rat and that any suppression of the tonic inhibitory influence on PRL secretion can reveal its existence. In this study, the effects of the DA-lowering stimulus of suckling was investigated at different times of day in lactating rats. In addition, the pattern of PRL secretion in freely lactating rats throughout a 24-hour period was studied. Female rats were separated from their pups for 6 h prior to reunion at either 03.00 (coincident with the N component), 12.00 (control) or 17.00 h (coincident with the D component) at various stages of lactation. Blood samples were collected from intra-atrial cannulae immediately before separation of pups and dams, immediately before reunion of pups and dams (0 time), and 15, 30, 60 and 120 min following reunion of pups and dams. Four days following parturition, dams suckled at either 03.00 or 17.00 h secreted significantly greater PRL than rats suckled at 12.00 h. Peak levels of PRL were 60-, 90- and 25-fold greater than 0 time levels, at 03.00, 17.00 and 12.00 h, respectively. Ten days following parturition, dams suckled at either 03.00 or 17.00 h secreted significantly greater PRL than those rats suckled at 12.00 h. Peak levels of PRL were 170-, 190- and 100-fold greater than 0 time levels, respectively. Eighteen days following parturition, dams suckled at 03.00 h secreted significantly greater PRL than rats suckled at either 12.00 or 17.00 h. Peak levels were 25-, 16- and 9-fold greater than 0 time levels, respectively. There was no difference in the PRL secretory responses of rats suckled at 12.00 and 17.00 h. There was no difference in the suckling-induced secretion of oxytocin and growth hormone between the times tested in rats in midlactation. The secretory profile of PRL in freely lactating rats throughout a 24-hour period was bimodal. Basal levels of PRL were evident from 09.00 to 15.00 h. A broad surge of PRL began at 16.00 h, peaked at 18.00 h and then slowly declined to basal levels by 02.00 h. Another brief surge of PRL peaked at approximately 06.00 h then quickly declined to basal levels. Taken together, these data are consistent with the presence of the ESR in lactating rats. The similarity of the periodicity and regulation of the bimodal daily rhythm of PRL present in freely lactating rats with surges of PRL in mated female rats suggest that this daily rhythm or PRL secretion is governed by the ESR. Lastly, these data are consistent with our original proposition that the ESR is continuously present in the female rat.