We have analysed early viral gene products expressed in adenovirus type 12 (Ad12)-infected cells as well as in two Ad12-transformed hamster cell lines, and Ad12-induced rat tumour cell lines by cell-free translation of virus-specific RNA which was selected by hybridization to cloned restriction endonuclease fragments of virus DNA. Proteins synthesized in vitro were analysed by one- and two-dimensional gel electrophoresis. It was found that RNA encoded by early region E1A directs the synthesis of at least eight polypeptides with apparent mol. wt. 38K, 36K, 30K, 28K, 26K, 25K, 24K and 22K. All these proteins are related to each other. E1B-specific RNA directs the synthesis of three proteins: 59K, 19K and 17K. Early region E2a codes for a 61K polypeptide which probably represents the single-strand DNA-binding protein of Ad12. RNA complementary to region E3 directs the synthesis of a 16K protein, and RNA transcribed from region E4 the synthesis of polypeptides with mol. wt. 20K, 18K and 11.5K. We have mapped a 67K polypeptide into the region within 11 to 28 map units (E2b). The analysis of proteins directed by virus-specific RNAs prepared from two Ad12-transformed hamster cell lines (T637, HA12/7) and one Ad12-induced rat tumour line (RBT12/3) showed that early regions E1 and E4 are expressed in all three Ad12-transformed cell lines. RNA transcribed from early regions E2 and E3 have been detected in lines T637 and RBT12/3. The virus RNA prepared from the Ad12-transformed cell lines directed synthesis of polypeptides with mol. wt. very similar to those of early virus proteins from infected cells. However, in all three Ad12-transformed cell lines mentioned above we have found RNAs which directed the synthesis of additional polypeptides of early regions E1 (34K) and E4 (25K, 24K) not detected in infected cells. The DNA sequence between 11 and 28 map units (coding for the 67K protein) is not expressed in the Ad12-transformed cells.