Previous experiments have demonstrated a link between transcriptional regulatory mechanisms acting during F9 cell differentiation and transcription control by the adenovirus E1A gene. We have isolated a number of differentiation-specific genes by cDNA cloning to determine if E1A exerts a coordinated control over differentiation specific gene expression. The mRNAs encoded by these cDNAs were undetectable or only barely detectable in undifferentiated cells but then rose in concentration upon differentiation. Analysis of transcription rates in isolated nuclei revealed that all but one of the genes was transcriptionally regulated during differentiation. Interestingly, alpha 2-type IV collagen expression was activated by a post-transcriptional mechanism since the gene was transcribed in both undifferentiated and differentiated cells whereas the cytoplasmic mRNA was undetectable in undifferentiated cells but rose in abundance in parallel with other regulated transcripts. Adenovirus infection of differentiated F9 cells reduced the cytoplasmic mRNA levels of each of the differentiation specific genes to near that found in the undifferentiated cell. Of those genes that were transcriptionally activated by differentiation, adenovirus infection specifically inhibited transcription. In contrast, although the alpha 2 collagen mRNA levels were reduced by adenovirus infection similar to the other mRNAs, the control was post-transcriptional since transcription of the gene was unaffected. Thus, the mechanism for loss of gene expression mediated by E1A reflects the mechanism by which the gene was activated during differentiation. Based on these results we suggest that E1A controls the expression of the F9 cell phenotype by targeting a regulatory activity acting early in the differentiation program.