Cellular proteins of 300, 107, 105, 68 and 65 kDa have previously been shown to associate specifically with the early region 1A (E1A) proteins of human adenovirus type 5. In the present study we report that, to varying degrees, these proteins also were capable of binding to E1A products produced in Escherichia coli from plasmids carrying cDNAs corresponding to the 1.1- and 0.9-kb E1A mRNAs. When these purified E1A proteins were mixed in solution with extracts from mock-infected human cells, the 68- and 65-kDa species bound very efficiently to the 1.1-kb mRNA product and somewhat less so with that of the 0.9-kb mRNA. The 107-, 105-, and 300-kDa species bound poorly, if at all, to both E1A products. Using the E1A 1.1-kb mRNA product which had been covalently attached to Sepharose beads, the 68-, 65-, and 300-kDa species bound efficiently, and binding of protein which migrated in SDS gels in the region of the 107- and 105-kDa species was also observed. In addition to these proteins, several other cellular polypeptides of 30, 33, 75, 95, 150, 180, and greater than 300 kDa were shown to bind to E1A-Sepharose and thus may also be E1A-binding proteins. The present data confirm the specificity of the previously identified cellular proteins for E1A products and show that binding of the 300-, 65-, and 68-kDa species does not require the presence of any other viral polypeptide. In contrast, the inefficient binding of the 107- and 105-kDa species to Escherichia coli-expressed E1A protein may suggest that these interactions require either eukaryotic-specific post-translational modifications of the E1A protein, or the presence of additional Ad5 gene products.