When applied topically to the skin twice at a 48-h interval or thrice at 24-h intervals, 17 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 mumol of A23187 or ionomycin induce the same 3-fold increases of hydroperoxide (HPx) production in mouse epidermis. In contrast, these doses of Ca2+ ionophores applied once or twice at a 48-h interval produce only 3-8% of the 16- or 34-fold inductions of epidermal ornithine decarboxylase (ODC) activities caused by similar TPA treatments. However, these various Ca2+ ionophore treatments mimic entirely the stimulatory effects of TPA on epidermal DNA synthesis at 16 h and produce from 30 to 70% of the DNA responses to TPA at 32 h. Interestingly, the Ca2+ ionophore and TPA treatments applied thrice at 24-h intervals still produce above maximal or submaximal DNA responses, in spite of their very weak ODC-inducing activities or refractoriness against ODC induction. Treatment with alpha-difluoromethylornithine plus methylglyoxal bis-(guanylhydrazone) (1.25 mumol each), which inhibits the activity of the polyamine-synthesizing enzymes, does not block the HPx and DNA responses to TPA. Conversely, 1.6-25-nmol doses of fluocinolone acetonide inhibit both TPA-induced HPx production and DNA synthesis, without affecting ODC induction. The results suggest that the magnitudes of Ca2+ ionophore- and TPA-induced DNA synthesis may be linked to HPx production rather than ODC induction. Each of these three responses appears to be essential but not sufficient for tumor promotion. A23187 may be a poor or incomplete skin tumor promoter because it lacks sufficient ODC-inducing activity and cannot fully maintain the prolonged stimulation of DNA synthesis required for hyperproliferation.