Early region 3 of the adenovirus type 2 genome encodes three proteins with molecular weights of 16,000, 14,500, and 14,000 (E2/16, E3/14.5, and E3/14). The E3/16 protein is the precursor to the E3/19 glycoprotein and is around 1500 daltons larger than the unglycosylated E3/19O protein. The E3/14.5 and E3/14 proteins are structurally related to each other but different from E3/16. Three mRNA species were identified for E3/16; all have common 5' ends with the same spliced region but with different 3' ends. E3/14 was translated from a 13S mRNA with the same 5' structure as the E3/16 mRNA but followed by a second spliced region with a different 3' end. A partial amino acid sequence was determined for E3/16 after radioactive labeling in vitro and this sequence can be aligned with a known DNA sequence. It contains a hydrophobic signal sequence, two presumptive glycosylation sites, and a hydrophobic region close to the COOH terminus.