Topical application of 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2D3], an active form of vitamin D3, markedly inhibited induction of ornithine decarboxylase caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) and teleocidin B in mouse skin. The degree of inhibition was dependent on the dose and time of application of 1 alpha, 25(OH)2D3. Application of 1 micrograms 1 alpha, 25(OH)2D3 within 30 min before or after treatment with 10 micrograms TPA caused about 72% inhibition of ODC induction at 4 hr. Similar degrees of inhibition were obtained with dose ratios of 1 alpha, 25(OH)2D3 to TPA of 1:3, 1:10, and 1:30. The dose required for 50% inhibition was 0.063 micrograms, or 0.15 nmol, which is about one-half that of retinoic acid, a known inhibitor of induction of ODC activity by TPA. 1 alpha, 25(OH)2D3 had a specific inhibitory effect, in which 100 times higher doses or more of other derivatives of vitamin D3, such as 1 alpha-hydroxyvitamin D3, 25-hydroxyvitamin D3, 24R,25-dihydroxyvitamin D3, and vitamin D3, were required to inhibit ODC induction by TPA. 1 alpha, 25(OH)2D3 did not inhibit epidermal hyperplasia induced by TPA.