Short term assays for potential carcinogenic factors must take into account the fact that carcinogenesis is usually a multistep process and that most human cancers probably result from a complex interaction between multiple factors, both environmental and endogenous. Thus, although extremely valuable, simple in vitro mutagenesis assays have major limitations. Cell culture studies with TPA and other phorbol esters provide clues to tumor promotion and the multistep nature of carcinogenesis. These effects can be divided into three categories: 1) mimicry of transformation in normal cells, and enhancement of transformation by chemical carcinogens or oncogenic viruses; 2) modulation (inhibition or induction) of differentiation; 3) membrane and receptor effects. Current evidence suggests that TPA acts by binding to specific high affinity cell surface membrane receptors and that this leads to alterations in the composition of membrane phospholipids. Presumably, these changes in the lipid matrix of cell membranes produce signals or mediators which lead to the subsequent cytoplasmic and nuclear effects of TPA. Thus, whereas the critical target in the action of initiating carcinogens appears to be cellular DNA, the critical target of the phorbol ester tumor promoters appears to be cell membranes. As a unifying concept of two-stage carcinogenesis, we postulate that during the initiation phase in carcinogenesis the covalent binding of carcinogens in DNA induces abberations in the commitment of the target cells. We believe that this involves highly ordered genetic events, for example, gene transpositions, rather than random point mutations. Certain oncogenic viruses achieve a similar effect thru integration of exogenous DNA sequences into inappropriate sites in the host genome. Tumor promoters, via their effects on growth, gene expression and differentiation, enhance the selective outgrowth of these initiated cells and induce them to express their newly acquired by previously dormant committed state. Rapid in vitro assays for the detection of the phorbol ester tumor promoters and for synergistic interactions between oncogenic viruses and chemical now exist. However, simple assays for other classes of tumor promoters and cofactors remain to be developed.