A number of chemicals which promote tumorigenesis in vivo have been observed to inhibit metabolic cooperation between 6-thioguanine-resistant (TGR) and sensitive (TGS) Chinese hamster lung V79 cells. The apparent correlation between an inhibition of metabolic cooperation in V79 cells in vitro and promotion of oncogenesis in vivo has led to the suggested utilization of the assay as a screen for tumor promoters. However, many features of the V79 metabolic cooperation assay which would provide for an improved understanding of the usefulness and limitations of the assay have not been well characterized. A number of experimental parameters involved in the metabolic cooperation assay were examined with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA inhibited metabolic cooperation between V79 cells in a dose-dependent manner, and thereby increased the recovery of TGR cells co-cultured with TGS cells in the presence of 6-thioguanine. Approximately 90% recovery of TGR cells was achieved at TPA concentrations of 1--1000 ng/ml, a 9- to 11-fold higher yield than the average background recovery of 8--10% in acetone-treated cultures. The TPA-induced inhibition of metabolic cooperation was observed to be transient. Pretreatment of TGS and TGR cells with TPA for 12 h or more prior to 6-thioguanine addition resulted in no inhibition of metabolic cooperation. It was further determined that the presence of TPA was required for only 1 h to maximally inhibit metabolic cooperation, a significantly reduced period of exposure relative to the originally proposed 4 day exposure. Technical grade dinitrotoluene (DNT), 2,4-DNT and 2,6-DNT which have demonstrated promoting activity in rat liver did not increase the recovery of TGR cells. However, the solvent dimethylsulfoxide (DMSO) at concentrations ranging from 1.0 to 2.5% increased the recovery of TGR cells in a dose-dependent manner. The short-lived effect of TPA suggests that inhibition of metabolic cooperation may not bear a mechanistic relationship to tumor promotion. The inhibition of metabolic cooperation by DMSO, the requirement for only short-lived reductions in metabolic cooperation for maximal TGR cell recovery, and the lack of inhibition by DNT suggests that caution should be exercised when interpreting the results of this bioassay.