We have further analyzed structure and expression of the E1 region of the transformation-defective adenovirus 12 (Ad12) host range mutant CS-1. Using cDNA polymerase chain reaction analysis, the E1a region was found to give raise to five transcripts analogous to the Ad12wt 13S, 12S, US, 10S species and the normal 9S mRNA. Due to loss of a splice acceptor site at position 852, the Ad12wt-specific 9.5S transcript cannot be synthesized by the mutant CS-1. The fact that the virus is, however, completely viable indicates that this mRNA is dispensable for lytic growth of Ad12. Besides the deletions in E1a described previously, a second deletion of 31 base pairs was found in the E1b gene. It affects the start site of the E1b-specific mRNAs and destroys or eliminates the AUG sequence for the E1b 19-kD protein. As shown earlier, normal E1b mRNA expression is found in mutant-infected cells, but no 19-kD E1b protein was immunoprecipitated by an E1-specific rat tumor serum.