Early region 1A (E1A) of human adenovirus type 5 (Ad5) produces five mRNAs that encode proteins of 55, 171, 217, 243, and 289 residues. We have shown previously that the major products of 289 and 243 residues are phosphorylated at a minimum of three sites of which one, Ser-89, is located in the amino terminal half of the protein. In the present report we show that these E1A proteins are also phosphorylated at a second site in this region located at Ser-96. The 171 and 217 residue E1A species were also tentatively identified and, as predicted, neither contained the Ser-89 or Ser-96 sites but both appeared to be phosphorylated at the same sites as 289R and 243R toward the carboxy terminus. Studies with mutants in which Ser-89 or Ser-96 were converted to alanine residues indicated that phosphorylation of Ser-89 but not Ser-96 induces the major shift in gel mobility of E1A products. However, neither site appears to be of major importance in the regulation of E1A-mediated transactivation or transformation.