Transformation suppressor Pdcd4 is downregulated in transformed (Tx) mouse epidermal JB6 RT101 cells relative to transformation-resistant (P-) and susceptible (P+) variants. Whether Pdcd4 downregulation is necessary not only to induce transformation but also to maintain tumor phenotypes has not been determined previously. In the present study, overexpression of Pdcd4 cDNA in stably transfected RT101 cells resulted in 40% fewer anchorage-independent colonies that were smaller in size than the vector control colonies, indicating that elevated Pdcd4 expression is sufficient to suppress tumor phenotype. Transient transfection of Pdcd4 expression plasmid and 4 x AP-1 reporter gene showed that activation of AP-1-dependent transcription was inhibited by Pdcd4 expression in a concentration-dependent manner. In contrast, Pdcd4 did not inhibit serum response element-dependent transcription, indicating specificity. In a Gal4 fusion assay, Pdcd4 specifically inhibited activation of c-Jun and c-Fos activation domains, but did not inhibit activation of JunB, JunD, Fra-1, or Fra-2. Gel mobility shift assay demonstrated that c-Jun is the major component detected in the AP-1 complex in RT101 cells. Previous studies suggested that AP-1 activity is required for maintaining the transformed phenotype in RT101 cells. Thus, Pdcd4 suppresses tumor phenotype by inhibiting AP-1-dependent transcription, possibly through inhibiting c-Jun and c-Fos activation.