JunB, a member of the jun gene family of transcription factors, is distinguished from c-Jun by its differential activity on certain arrangements of promoter regulatory elements and the ability of JunB to inhibit the action of cJun in both transforming and trans-activating assays. We have tested the potential negative regulatory role of JunB during the retinoic acid induced differentiation of F9 murine embryonal carcinoma cells. Constitutive expression of high levels of JunB in F9 cells failed to inhibit the differentiation dependent induction of c-Jun or the coincident expression of differentiation markers keratin 8 and 18, tissue plasminogen activator, and laminin B1. Among these marker genes, keratin 18, has been shown to contain an AP-1 binding site, TGA(C/G)TCA, which is essential for high level, differentiation dependent expression and which is transactivated by Jun and Fos proteins. These results suggest that JunB does not play a major negative or positive regulatory role during the retinoic acid induced differentiation of F9 cells.