To determine whether tumor necrosis factor-alpha (TNF alpha) plays a physiological role in normal mammary gland development, TNF alpha and TNF receptor expression were measured in epithelial cells (MEC) isolated from mammary glands of virgin, pregnant, lactating, and postlactational (day 7 of involution) rats. TNF alpha messenger RNA (mRNA) increased significantly during pregnancy, then decreased during lactation and involution. The 26-kDa transmembrane form of TNF alpha protein, undetectable in MEC from virgin rats, increased throughout pregnancy and lactation, and disappeared during involution. In contrast, p55 TNF receptor (TNFR) mRNA levels peaked in early lactation and declined thereafter, whereas p75 TNFR mRNA levels rose steadily through lactation. Using agonistic antibodies specific to either TNFR, the individual roles of each TNFR were investigated in MEC in primary culture. The p55 TNFR was found to be the sole mediator of TNF alpha-induced proliferation. Intriguingly, the two receptors were found to have opposing effects on functional differentiation (casein accumulation), with inhibition occurring through the p55 receptor and stimulation through the p75 receptor. Taken together, these results suggest that TNF alpha plays a role in the growth and development of the mammary gland. In addition, both TNF receptors are important for TNF alpha function and may mediate different effects.