Prolactin (PRL) serves an important luteotrophic function in the rat during early pregnancy, expressed as a nocturnal surge in the early morning and a diurnal surge in the late afternoon. Several areas of the hypothalamus, including the preoptic area (POA), the suprachiasmatic nucleus (SCN) and the ventromedial and dorsomedial nuclei (VM-DM) have been implicated in PRL surges. We investigated the temporal relationship between neuronal activity as measured by c-Fos immunocytochemistry in these areas and PRL secretion during early and late pregnancy. Brains were collected at nine time points (24:00, 02:00, 04:00, 06:00, 10:00, 14:00, 16:00, 18:00 and 20:00 h) on days 6–7 and three time points (02:00, 14:00 and 18:00 h) on days 14–15 of pregnancy. Plasma PRL levels determined by radioimmunoassay revealed two surges with peaks at 02:00 and 18:00 h and a trough at 14:00 h on days 6–7, which were absent on days 14–15 of pregnancy. The number of neurons expressing c-Fos in the anterior medial preoptic nucleus, the medial preoptic area and the medial preoptic nucleus, but not the anteroventral preoptic nucleus of the POA, and the VM-DM, showed a semicircadian rhythm which was maximal at 02:00 h or/and 04:00 and 18:00 h and reached the lowest value at 14:00 h, in parallel with the PRL surges in early pregnancy. However, the temporal pattern of c-Fos in these areas was reversed during late pregnancy, with a peak at 14:00 h and low levels at 02:00 and 18:00 h. PRL surges were absent and levels were uniformly low during these times. Neuronal activity in the SCN did not show any correlation with PRL surges. The dorsomedial subdivision of the SCN showed high neuronal activity during the daytime in both stages of pregnancy. Neuronal activity in the ventrolateral subdivision of the SCN was high during the nighttime in early pregnancy, however it exhibited high levels during the daytime in late pregnancy. These results suggest that the two daily surges of PRL secretion during the first half of pregnancy might be related to the temporal rhythm of neuronal activity in the POA and the VM-DM, and a major change in the pattern of neuronal activity in these hypothalamic areas might result in termination of the PRL surges at midpregnancy.