Activin and inhibin, two closely related protein hormones, are members of the transforming growth factor beta (TGF beta) superfamily of growth factors. Activin and TGF beta have been associated with mouse mammary gland development and human breast carcinogenesis. TGF beta expression in the mammary gland has been previously described, and was found to be expressed in nonparous tissue and during pregnancy, down-regulated during lactation, and then up-regulated during involution. The expression pattern of activin subunits, receptors and cytoplasmic signaling molecules has not been thoroughly described in post-natal mammary gland development. We hypothesize that activin signaling components are dynamically regulated during mammary gland development, thereby permitting activin to have distinct temporal growth regulatory actions on this tissue. To examine the activin signal transduction system in the mammary gland, tissue from CD1 female mice was dissected from nonparous, lactating day 1, 10, and 20 and post-weaning day 4 animals. The expression of the activin receptors (ActRIIA, ActRIIB and ActRIB), the inhibin co-receptor (betaglycan), and ligand subunit (alpha, beta A and beta B), mRNA was measured by semi-quantitative RT-PCR in these tissues. In addition, the cellular compartmentalization of the activin signaling proteins, including the cytoplasmic signaling co-activators, Smads 2, 3 and 4, were examined by immunohistochemistry. Generally, mRNA abundance of activin signaling components was greatest in the nonparous tissue, and then decreased, whereas protein immunoreactivity for activin signaling components increased during lactation and decreased during involution. The alpha-subunit protein was detected in nonparous and lactating day 1 tissue only. Importantly, Smad 3, but not Smad 2, was detected in epithelial cell nuclei during all time points examined, indicating that activin signaling is mediated by Smad 3 at these times. These findings suggest that activin's growth regulatory role during lactation may be distinguished from that of TGF beta during post-natal mammary development. Future studies will focus on determining the exact role this ligand plays in mammary tissue differentiation and neoplasia.