The aliphatic diesters of phorbol (eq. 12-O-tetradecanoylphorbol-13-acetate (TPA) are extremely potent tumor promoters in mouse skin and potent mitogenic agents in mammalian cells in vitro. In mouse epidermal cells in culture, TPA induces a number of changes in morphology, growth, and biochemical parameters. Some changes include increased phospholipid synthesis, increased ornithine decarboxylase activity, increased prostaglandin E2 synthesis, increased DNA synthesis, change in morphology to a more fibroblastic appearance, and changes in keratinization. This investigation of the mode of action of TPA in epidermal cells will have two objectives: 1) to further understand the mechanism of tumor promotion in a known target cell; and 2) to elucidate the mechanism by which TPA triggers cell proliferation and/or alters differentiation in a target tissue. The initial approach will entail modifying the aliphatic ester groups to contain an affinity label and a photoaffinity label, thereby facilitating location of the phorbol diester on or within the cell and allowing characterization of the site(s). The negative controls for these probes will be synthetic, nonpromoting, hyperplastiogenic phorbol diesters. We will attempt to define the effects of the nonpromoting hyperplastiogenic phorbol diesters on in vitro cell proliferation and to ascertain if the effects on cell proliferation and differentiation have a similar origin. Our results may clarify the role of cell proliferation in tumor promotion in epidermal cells and may lead to a more precise bioassay for epidermal tumor promoters.