To investigate possible mechanisms through which 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) affects cell proliferation and differentiation, we have studied the effects of 1,25-(OH)2D3 on the binding and mitogenic activity of epidermal growth factor (EGF) in RCJ 1.20 cells, an established, non-tumorigenic cell line derived from 21-day-old fetal rat calvaria. 1,25-(OH)2D3 caused a dose- and time-dependent 2- to 3-fold increase in the number of receptors for EGF. The 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 metabolites of vitamin D3 were ineffective in eliciting changes in EGF binding. Saturation and Scatchard analyses indicated that an increase in available unoccupied high affinity EGF binding sites was responsible for the 1,25-(OH)2D3-induced EGF binding. In addition, 1,25-(OH)2D3 enhanced EGF-dependent growth of RCJ 1.20 cells in soft agar. The potentiation of EGF effects on RCJ 1.20 cell growth by 1,25-(OH)2D3 may be related to the 1,25-(OH)2D3 regulation of EGF binding. However, the induction of anchorage-independent growth by 1,25-(OH)2D3 appears to be due to the stimulation of transforming growth factor beta-like activity. These results provide a possible explanation for the mechanism whereby the effects of 1,25-(OH)2D3 on cell proliferation and bone metabolism may be mediated.