Middle-T antigen is the oncogenic protein of Polyomavirus and associates with several cellular enzymes involved in signal transduction, e.g., Src tyrosine kinases, phosphatidylinositol 3-kinase (PI 3-kinase), protein phosphatase 2A (PP2A), and Shc, an SH2 domain-containing adapter protein. We have shown earlier that middle-T is a target of a cell cycle-regulated serine/threonine-specific kinase, presumably p34cdc2. Phosphorylation of middle-T by p34cdc2 results in increased apparent M, weight of the protein on SDS-polyacrylamide gels. Two threonine residues in positions 160 and 291, respectively, were identified in the middle-T sequence as putative targets of a cyclin-dependent kinase. Replacement of threonine 160 by alanine resulted in a transformation-defective mutant protein that was still capable of forming all the complexes with cellular proteins, suggesting that additional characteristics of middle-T are required for cell transformation. In the present study we report that the defect of the T160A middle-T mutant is compensated by mutations introduced into a domain encompassing amino acids 253 to 302. In particular, mutating serine 283, a canonical phosphorylation site for a cyclin-dependent kinase, to an alanine residue rendered the T160A middle-T mutant wild type. Based on these results we suggest that cell cycle-specific phosphorylation of specific serine and threonine residues by cyclin-dependent kinases regulates middle-T function.