Changes in prolactin (PRL)-, growth hormone (GH)- and PRL/GH-containing cells in the anterior pituitary of pre- and postnatal male and female rats were determined using immunocytochemistry with double fluorescent antibodies. The pituitary glands from a fetus on Day 20 of gestation and pups on Days 0, 1, 4, 7, 12, and 20 of the postnatal period with sex distinction were monodispersed and subjected to immunocytochemistry. Following immunostaining, the three types of cells described above were counted (i.e., only PRL-, only GH-, and both PRL and GH-positive cells in a visual field of microscope). Anterior pituitaries and blood were obtained from pups on Days 0, 1, 4, 7, 12, and 20 after birth and pooled with sex distinction, and bioactive PRL levels in the anterior pituitary and serum were measured using an Nb2 rat lymphoma cell bioassay. Double fluorescent-labeled immunocytochemistry was able to distinguish reliably between PRL-, GH-, and PRL/GH-containing cells. The PRL cells increased rapidly after parturition to 4 days of age and after 12 days of age in both genders. The latter increase was involved in the remarkable increase of PRL/GH-containing cells, which were first found at term and remained at low levels until 12 days of age. The weight and PRL level of the anterior pituitary increased in both sexes throughout the lactational stage, but the levels in female rats at 20 days of age were significantly greater than those of male rats. Serum PRL concentration remained at low levels except on Day 0. The present results demonstrate that the development of PRL-containing cells in postnatal rats during the lactational period is activated with two distinct stages, at term to Day 4 and after Day 12, and the development in the latter stage is involved with sexual dimorphism.