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The in vitro V79/metabolic cooperation assay measures the extent of gap-junctional transfer of metabolites from wild-type to mutant V79 cells. The assay is currently being explored as a short-term test to screen for tumor promoting chemicals, many of which inhibit metabolic cooperation. In this study, the assay was used to determine whether chemical interactions affect detection of tumor promoters in mixtures and to investigate types of interactions that may occur between chemicals. Several two-chemical mixtures were examined. The effects of phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate, two inhibitors of metabolic cooperation that operate through the same receptor-mediated pathway, were additive at concentrations below the maximally effective concentrations of either. A summation effect was observed in mixtures of two other inhibitors of metabolic cooperation, the pesticide aldrin and the principal metabolite of sodium cyclamate, cyclohexylamine. Synergistic effects were noted when PMA was combined with either aldrin or cyclohexylamine, demonstrating that chemicals in a mixture may yield a much stronger response than expected based on individual chemical exposures. Interactions were also examined between PMA, aldrin, cyclohexylamine and 2,4-diaminotoluene, a chemical that appears to enhance metabolic cooperation. 2,4-Diaminotoluene reversed effects of all inhibiting chemicals to some extent, although the pattern of response was different for each combination. In the most dramatic case, the powerful tumor promoter PMA was completely masked by 2,4-diaminotoluene. These results suggest that the V79/metabolic cooperation assay must be applied with caution in mixture testing because detection of tumor promoting chemicals can depend on other chemicals present.