Cytokines such as tumor necrosis factor α (TNFα) have been implicated in amniotic fluid infections and preterm and term labor. The underlying mechanisms are incompletely understood. In some smooth muscle cells, TNFα affects function of the β-adrenergic/adenylyl cyclase pathway. The present study was performed to examine the effects of chronic TNFα exposure on adenylyl cyclase activity in cell cultures of human myometrium. Chronic TNFα exposure led to a dose- and time-dependent increase in basal-, GTP-, NaF-, and forskolin-stimulated adenylyl cyclase (AC) activity. The increase in AC activity was not mediated by changes in the expression of the heterotrimeric G proteins G_sα or G_iα as determined by immunoblotting. In addition, increases in AC activity occurred in the presence of indomethacin, indicating that these changes were not provoked by TNFα-induced changes in prostaglandin production. The present results suggest that TNFα-induced increases in AC activity in human myometrial cells obtained from the lower uterine segment occur at the level of G-protein/AC interaction or at the level of the AC enzyme itself.