Adenovirus 12S E1A gene represses differentiation of F9 teratocarcinoma cells. The F9 teratocarcinoma cell line differentiates in vitro after treatment with retinoic acid and cAMP and has been a widely used model system for the study of the molecular events that are responsible for cellular commitment and differentiation during early development. Previous experiments have suggested intriguing parallels between the control of gene expression during F9 cell differentiation and the regulation of gene expression by adenovirus E1A. Transfection of a 12S E1A-expressing plasmid into terminally differentiated, nonproliferating F9 cells generates, at high frequency, colonies of dividing cells, each of which expresses E1A. Cell lines established from these colonies proliferate in the presence of retinoic acid and have lost the fully differentiated phenotype as characterized by the absence of expression of a series of differentiation-specific genes. We conclude that expression of the viral 12S E1A gene product interferes with retinoic acid-induced F9 cell differentiation. Moreover, the results suggest that the differentiation process, as defined by markers of terminal differentiation, may not be a permanent event but can be reversed by E1A expression. 