The 12 S and 13 S E1A cDNAs from both the Adenovirus (Ad) nononcogenic type 5 and the oncogenic type 12 were overexpressed in an insect cell/baculovirus system. Upon infection of Spodoptera frugiperda cells, the production of E1A proteins reached a level of about 15 micrograms/10(6) cells. The E1A proteins are highly soluble and apparently are processed authentically. They are readily recognized by various antibodies and display phosphorylation patterns similar to those of E1A proteins synthesized in mammalian cells. Single-step immunoaffinity chromatography was used to purify the Ad5 E1A proteins to near homogeneity under nondenaturing conditions. The Ad5 and Ad12 E1A proteins are able to form complexes with the retinoblastoma susceptibility gene product (Rb) and other cellular proteins. Interestingly, the presence of a cellular extract seems to be a prerequisite for association between highly purified E1A and Rb polypeptides.