The activity of the A14 and A12 hypothalamic dopaminergic (DAergic) neurons were characterized throughout a day of pseudopregnancy to establish their relationship to the generation of PRL surges during that time. Double-label immunocytochemistry was performed using fos-related antigens as markers of neuronal activity, and tyrosine hydroxylase antibody to identify DAergic neurons of the periventricular nucleus and the arcuate nucleus (ARN). Dopamine concentrations were measured with HPLC with electrochemical detection from micropunched samples to approximate synthetic activities of A14 and A12 neurons. Serum PRL levels were determined by RIA. On the fifth and sixth day of pseudopregnancy, five rats/time point were killed at 1100 h, 1300 h, 1500 h, 1800 h, 2100 h, 2400 h, 0300 h, and 0600 h. When the serum PRL was low, the incidence of fos-related antigens/tyrosine hydroxylase double-labeled neurons was high, and it decreased twice a day by 1500 and 0300 h in the periventricular nucleus, rostral ARN, dorsomedial subpopulation of the middle ARN, and in the caudal ARN, corresponding to the initiation of PRL surges. Concentrations of DA were high in these areas, and decreased before PRL surges in agreement with the immunocytochemical data. These findings suggest a semicircadian rhythm in the activities of A14 and A12 neurons which, in concert with PRL releasing factors, contribute to the generation of PRL surges in pseudopregnant animals.