The effect of different phorbol esters and of mechanical treatment on the activity of ornithine decarboxylase in mouse epidermis in vivo was investigated. The strong promoter 12-O-tetradecanoylphorbol-13-acetate as well as the weak promoters phorbol dibenzoate and the 12-O-tetradecanoylphorbol-13-acetate analog 12-O-tetradeca-2-cis, 4-trans-6,8-tetraenoylphorobol-13-acetate strongly increased the activity of the enzyme and the intraepidermal level of putrescine, with a maximum at 5 hr after application, when applied in doses which evoke comparable proliferative and irritant responses in skin. The hyperplasiogenic but nonirritant and almost nonpromoting 4-O-methyl ether of 12-O-tetradecanoylphorbol-13-acetate did not show such effects. Mechanical removal of the uppermost horny layer led to a considerable increase of ornithine decarboxylase activity after 4 to 8 hr, while skin massage showed only a minute effect under conditions in which both treatments exhibit about the same mitogenic efficiency. Neither manipulation promotes tumor development. After skin massage, the induction of ornithine decarboxylase was influenced neither by treatments which alter the cyclic adenosine 3',5'-monophosphate level in epidermis (inhibition of phosphodiesterase, beta-adrenergic stimulation, and injection of dibutyryl cyclic adenosine 3',5'-monophosphate) nor by injection of epidermal G1 chalone. The results indicate that no clear-cut correlation exists between epithelial cell proliferation, development of hyperplasia, and tumor promotion on the one hand and an activation of epidermal ornithine decarboxylase on the other.