During pregnancy, in response to rising levels of estrogen and progesterone, mammary epithelial cells proliferate and differentiate, giving rise to lobuloalveolar structures from which milk is secreted during lactation. Progesterone, in conjunction with estrogen, also promotes mammary epithelial growth during puberty, but unlike development during pregnancy, this results in ductal rather than secretory structures. Recent studies using mice lacking progesterone receptors indicate that these receptors are essential for secretory development. This suggests that during different phases of normal mammary growth and differentiation, progesterone receptors may mediate their effects through different mechanisms, depending on the physiological state of the animal. Therefore, as a prerequisite for understanding the role of progesterone in normal mammary development, we have investigated the spatial and temporal distribution of progesterone receptor-containing cells. In the mammary epithelium of prepubertal mice, high levels of progesterone receptor mRNA and protein are present in the actively growing end buds and ductal branches as well as in the mature duct. Receptor protein was found in some but not in all epithelial cells, and these cells were characterized by large, round, or oval nuclei containing diffuse chromatin. Cytoplasmic and nuclear immunostaining were observed; interestingly, this staining occurred in separate cells that were in close proximity to each other. The relative proportion of cytoplasmic versus nuclear staining cells changed during pregnancy such that in this developmental state, the nuclear staining cells predominated. The potential significance of these observations as they pertain to normal mammary epithelial growth and differentiation are discussed.