17 beta-estradiol (E2), progesterone (P) and testosterone (T) were investigated for their effects on the proliferation and differentiation of primary rat calvarial osteoblasts in vitro. Rat calvarial osteoblasts were cultured with 10(-10) mol/L E2, 10(-9)-10(-6) mol/L P and 10(-10) mol/L T for 20 days. Cell proliferation was determined by 3H-thymidine incorporation and cell counting. Cell differentiation was examined by measuring alkaline phosphatase (ALP) activity, osteocalcin gene expression and production, bone nodule formation in different periods of culture. Our results showed that no effect of three sex hormones was observed on cell proliferation, but, the responses of cell differentiation to the hormones were more or less different. Among these three hormones used in this study, P appeared to have multi-stimulating effect on cell differentiation. Effect of T seemed not so significant as that of P on cell differentiation. Although ALP activity and osteocalcin production were increased significantly by T, it had slight effect on osteocalcin mRNA and bone nodule formation. Besides, E2 did not demonstrate any effect on cell differentiation. It is concluded that the proliferation of rat calvarial osteoblasts was independent of the presence of sex hormones. However, the differentiation of these cells were stimulated in different levels and to different extent either by P or T. P appeared to be a multi-stimulator on differentiation of such cells.