Both TGF-β and 1,25-dihydroxy-vitamin-D3 (1,25(OH)2D3) have been reported to decrease the proliferation of normal human keratinocytes. The effect and expression of TGF-β in keratinocytes treated with 1,25(OH)2D3 was investigated. Human keratinocytes were grown in the presence of various concentrations of TGF-β and/or 1,25(OH)2D3 prior to enumeration. TGF-β, alone, has a half maximal dose of inhibition (ED50) of approximately 750 pg/ml after seven days in culture in Keratinocyte Growth Medium (KGM®; Clonetics) supplemented with 1.5 mM calcium. When 1,25(OH)2D3 (10−7M) was also added to cultures with various concentrations of TGF-β, the ED50 shifted an average of 2-fold less. The presence of TGF-β (10 pg/ml) augmented the potency of 1,25(OH)2D3 by at least 10-fold. In keratinocyte cultures, the antiproliferative effect of the two compounds together is synergistic. In keratinocytes grown for 1 week in the presence of 1,25(OH)2D3 at 10−6M, the TGF-β1 message increased approximately 5-fold. An increase is detected within 2 hours of exposure to 1,25(OH)2D3. There was only a 50% increase in the levels of TGF-β2 and no detection of TGF-β3. When keratinocyte cultures were treated with 1,25(OH)2D3 and neutralizing antibodies to TGF-β, the induced-antiproliferative activity was blocked by more than 50%. The keratinocytes produced more active than latent TGF-β after growth with high doses of 1,25(OH)2D3. © 1992 Wiley-Liss, Inc.