The JB6 mouse epidermal cell system has been extensively used as an in vitro model for the study of tumor promotion. The present study aimed to assess the relevance of monolayer measurements to the process of transformation, which is induced more efficiently under anchorage-independent (AI) conditions. Although it would be ideal to use identical con ditions for studying tumor promoter-induced transformation and biochemical and molecular events that may cause the process, it is not feasible in the case of soft agar conditions because cells cannot be readily recovered, in the present report, we used liquid medium over agar as an AI condition that permited efficient recovery of cells. Responses to tumor promoter have been compared with those in monolayer and semisolid agar. Results indicate that 12-O-tetradecanoyl- phorbol-13-acetate (TPA) induced similar magnitude concentration-dependent transformation of JB6 cells under both of the AI conditions, namely soft agar and over-agar. Under anchorage-dependent (AD) conditions of exposure to TPA, the transformation efficiency was much lower than that seen under Al conditions. Mechanical detachment of monolayer cells after 5-10 days TPA exposure enriched the transformed phenotype. Activator protein 1 transcrIptional activity measured at 12 h was induced equally under AD and AI conditions, and thus Is not an early limiting event that could explain the lower transformation efficiency seen under AD conditions. To summarize, the over-agar and monolayer assays described in this study can be considered valid for the study of early biochemical and molecular events relevant to the promotion of transformation measured in soft agar.