There is substantial evidence that the tumor promoter 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA) elicits enhanced arachidonic acid release and its metabolism to prostaglandins and lipoxygenase products in many cell types. The goal of this study was to determine whether 4 alpha-12-O-tetradecanoylphorbol-13-acetate (4 alpha TPA), a stereoisomer of TPA, can induce arachidonic acid release and whether it is by the same mechanism as release induced by TPA. The finding that 10 micrograms/ml 4 alpha TPA produces a response comparable with 1 microgram/ml TPA and with similar kinetics was unexpected. The mechanism mediating the TPA response appears to be the activation of protein kinase C (PKC), which subsequently results in phospholipase A2 activation. This is suggested by the observation that TPA-induced arachidonate release is inhibited 65% by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of PKC and that TPA completely down-regulates PKC. In addition, down-regulation or depletion of PKC by prior treatment with TPA results in a 75% loss of response to a second TPA treatment. In vitro activation of partially purified PKC could be demonstrated for TPA but not 4 alpha TPA. 4 alpha TPA thus appears to induce the release of arachidonate by a different but unknown mechanism. The 4 alpha TPA effect is not significantly reduced by the PKC inhibitor H-7, and no evidence of PKC activation or down-regulation was observed. Additionally, 4 alpha TPA is unable to "down-regulate" arachidonate release when the two-treatment protocol is used and the down-regulation of PKC by TPA has little effect on 4 alpha TPA-induced arachidonate release. Cycloheximide inhibited TPA-induced arachidonate release by 80% and 4 alpha TPA-induced release by 50%, indicating a partial requirement for protein synthesis for both phorbol esters. Actinomycin D, on the other hand, inhibited the TPA response by 70%, but enhanced the 4 alpha TPA response by 169%. When used at 10- or 100-micrograms doses, 4 alpha TPA was found to lack activity with respect to ornithine decarboxylase induction, oxidant production, hyperplasia, inflammation, and tumor promotion, suggesting that arachidonate release is not sufficient to induce these events. This may be related to the observation that with TPA the extent of arachidonate metabolism to prostaglandin E2 is four- to fivefold greater than occurred with 4 alpha TPA, even under conditions of equivalent arachidonate release.