Chimeric adenovirus type 5 (Ad5)/Ad12 early region 1A (E1A) genes were used to transform primary baby rat kidney cells in cooperation with Ad12 E1B, and the resulting cell lines were assayed for tumorigenicity in syngeneic rats. It was found that lines were nontumorigenic when transformed by hybrid E1A genes consisting of the amino-terminal 80 amino acids from Ad12 including conserved region 1 (CR1), with the remaining portion from Ad5. In contrast, cell lines transformed by hybrids containing Ad12 E1A sequences from the amino terminus to the leftmost border of CR3 or beyond were tumorigenic. To extend these results, sequences spanning CR2 and CR3 of Ad5 E1A were replaced with the homologous regions of Ad12 E1A and additional transformed cell lines were established. These lines were weakly-to-moderately tumorigenic, suggesting that Ad12 E1A sequences between CR2 and CR3 may be involved in tumorigenicity but are not the sole factors influencing it. Interestingly, examination of an E1A sequence alignment indicated that the region between CR2 and CR3 of Ad12 E1A is also conserved in the corresponding sequence of simian adenovirus type 7, which, like Ad12, is highly oncogenic. This region is characterized by the presence of a stretch of several alanine residues and is similar to a motif present in a number of proteins with transcriptional repression activity. The possibility that this region may influence tumorigenicity by means of a transcriptional regulatory mechanism is discussed.