We previously developed two models of human osteoblasts with distinct differentiation stages using cells derived from iliac crest trabecular bone explants cultured long term in the presence (HOB + DEX) and absence (HOB - DEX) of 10 nM dexamethasone (DEX) (Wong et al., J Bone Miner Res 1990;5:803). Using these models from 36 subjects aged 41-80 years, we examined the effects of 17 beta-estradiol (E2) on cell proliferation, osteocalcin (OC) production, alkaline phosphatase (ALP) and basal and parathyroid hormone (PTH)-stimulated adenylate cyclase activities, as well as the steady-state mRNA levels of ALP, collagen type I(COLL), OC, and receptors for E2 (ER) and PTH (PTHr). E2 alone had no effect on [3H]thymidine uptake in (HOB - DEX) cells but appeared to stimulate the uptake in (HOB + DEX) cells in a dose-dependent manner, with maximum effect at 10(-10)M (p < 0.05). However, in the presence of 10(-6)M PTH, E2 inhibited the uptake in (HOB - DEX) cells (ANOVA, KW = 18.95, p < 0.005) but stimulated the uptake in (HOB + DEX) cells (KW = 13.52, p < 0.025). E2 decreased the amount of osteocalcin in culture media from both (HOB - DEX) and (HOB + DEX) cells (p < 0.05). PTH alone or E2, alone or in combination with 10(-9)M PTH, had no effect on ALP activity in (HOB - DEX) cells. In contrast, in (HOB + DEX) cells, E2 + PTH but not E2 alone, had biphasic effects on ALP activity, with maximum stimulation observed at 10(-11) and 10(-10)M E2, and a return to basal levels at 10(-9)M E2. E2 decreased basal adenylate cyclase activities in a dose-dependent manner in (HOB + DEX) but not (HOB - DEX) cells (KW = 13.48, p < 0.05). In (HOB + DEX) cells, E2 had biphasic effects on PTH-stimulated adenylate cyclase activity, with significant stimulation observed at 10(-10)M (p < 0.05). While E2 had no significant effect on osteoblastic marker mRNA levels in (HOB - DEX) cells, it decreased osteocalcin and stimulated PTHr mRNA levels in (HOB + DEX) cells. Thus, in our human osteoblastic cell models, estrogen regulated metabolic function largely in the more differentiated cells, by modifying the effects of PTH.