The mechanism by which mechanical strain and estrogen stimulate bone cell proliferation was investigated using monolayer cultures of human osteoblastic TE85 cells and female human primary (first-passage) osteoblasts (fHOBs). Both cell types showed small but statistically significant dose-dependent increases in [3H]thymidine incorporation in response to 17β-estradiol and to a single 10-minute period of uniaxial cyclic strain (1 Hz). In both cell types, the peak response to 17β-estradiol occurred at 10−8-10−7 M and the peak response to strain occurred at 3500 microstrain (μϵ). Both strain-related and 17β-estradiol-related increases in [3H]thymidine incorporation were abolished by the estrogen receptor (ER) modulator ICI 182,780 (10−8 M). Tamoxifen (10−9-10−8 M) increased [3H]thymidine incorporation in both cell types but had no effect on their response to strain. In TE85 cells, tamoxifen reduced the increase in [3H]thymidine incorporation associated with 17β-estradiol to that of tamoxifen alone but had no such effect in fHOBs. In TE85 cells, strain increased medium concentrations of insulin-like growth factor (IGF) II but not IGF-I, whereas 17β-estradiol increased medium concentrations of IGF-I but not IGF-II. Neutralizing monoclonal antibody (MNAb) to IGF-I (3 μg/ml) blocked the effects of 17β-estradiol and exogenous truncated IGF-I (tIGF-I; 50 ng/ml) but not those of strain or tIGF-II (50 ng/ml). Neutralizing antibody to IGF-II (3 μg/ml) blocked the effects of strain and tIGF-II but not those of 17β-estradiol or tIGF-I. MAb αIR-3 (100 ng/ml) to the IGF-I receptor blocked the effects on [3H]thymidine incorporation of strain, tIGF-II, 17β-estradiol, and tIGF-I. HOBs and TE85 cells, act similarly to rat primary osteoblasts and ROS 17/2.8 cells in their dose-related proliferative responses to strain and 17β-estradiol, both of which can be blocked by the ER modulator ICI 182,780. In TE85 cells (as in rat primaries and ROS 17/2.8 cells), the response to 17β-estradiol is mediated by IGF-I, and the response to strain is mediated by IGF-II. Human cells differ from rat cells in that tamoxifen does not block their response to strain and reduces the response to 17β-estradiol in TE85s but not primaries. In both human cell types (unlike rat cells) the effects of strain and IGF-II as well as estradiol and IGF-I can be blocked at the IGF-I receptor.