Human adenovirus early region 1A (E1A) proteins act as transcriptional regulators and function in the control of DNA synthesis and cell transformation. Little is known about how these viral products are functionally regulated. E1A proteins of adenovirus serotype 5 (Ad5) are phosphorylated at several serine residues and previous studies had indicated that both Ser-89 and Ser-219 are substrates for one or more of the cdc2 family of cell cycle kinases. A second residue near the amino terminus, Ser-96, may also be a site. Although phosphorylation of Ser-89 causes a major shift in gel mobility, the effect on E1A biological activity is unclear. In the present studies we have shown by mutational analysis that phosphorylation at Ser-89 also regulates phosphorylation at Ser-96, suggesting that the gel mobility shift is the result of multiple phosphorylation events. Phosphorylation at Ser-89 did not seem to affect E1A-mediated repression of the simian virus 40 enhancer or trans-activation of the E3 promoter significantly, but it did appear to have a modest but significant effect on transformation of primary baby rat kidney cells.