Parathyroid hormone (PTH)-stimulated production of cAMP by intact human osteoblast-like SaOS-2 cells is diminished by pretreatment with 17 beta-estradiol (E2). The goal of the present study was to determine whether E2, affected adenylyl cyclase activity in cell membranes. Cells were deprived of steroid hormones for 24 h and then incubated with E2 (1 nM) or vehicle for 12 h. Cell membranes were prepared and incubated with [alpha-32P]-ATP in the absence or presence of agonist, and the amount of [32P]-cAMP produced was measured to quantify adenylyl cyclase activity. There was less cAMP produced by membranes from E2-treated cells in response to PTH, GTP gamma S, forskolin, and Mn2+. E2 had no effect on the amounts of Gs or Gi. We propose that pretreatment of SaOS-2 cells with E2 reduced membrane cyclase activity, at least in part, by actions on the PTH-sensitive adenylyl cyclase and/or the G protein-adenylyl cyclase complex.