The metabolism of [20-3H]-12-O-tetradecanoylphorbol-13-acetate ([3H]TPA) was studied in human and hamster cell cultures. Within 2 to 3 days after its addition to growing or confluent cultures of hamster embryo fibroblasts, no unchanged [3H]TPA remained in the medium as determined by thin-layer chromatography of the chloroform phase obtained by extraction of the medium with chloroform:methanol:H2O. In contrast, little or no metabolism of [3H]TPA occurred under identical conditions in cultures of human fibroblasts. The major metabolite formed from [3H]TPA in hamster cell cultures was [3H]phorbol-13-acetate. with both hamster and human cells, virtually all cell-associated radioactivity was unchanged [3H]TPA.