The purpose of this study was to investigate the relationships between macrophage production of TNF-alpha and female hormones. Northern blot hybridization experiments showed that the female sex steroid hormone, progesterone, decreases steady state levels of TNF-alpha mRNA in LPS-activated mouse macrophages (RAW 264.7 and ANA-1 cells) in vitro. The production of intracellular and secreted TNF-alpha protein, as determined by ELISA, was decreased in both progesterone- and dexamethasone-treated, LPS-stimulated macrophages. Estrogen had no effect on expression of the TNF-alpha gene in mouse macrophages and did not alter progesterone-mediated suppression. Additional experiments conducted to investigate the mechanism of action of progesterone showed that this hormone, like dexamethasone, elevates steady state mRNA levels of IkappaB alpha and increases the levels of IkappaB alpha protein that are translocated from the cytoplasm to the nucleus. Thus, progesterone is a potent inhibitor of steady state levels TNF-alpha mRNA and TNF-alpha protein production in activated macrophages and may achieve this result through effects on an inhibitor of NF-kappaB.