Full-length wild type and deletion mutant human androgen receptors (AR) were transiently expressed in monkey kidney COS cells to identify the phosphorylated amino acid residues. Phosphoamino acid analysis indicated serine (Ser) and threonine (Thr) residues as the major sites of phosphorylation. Both NH2- and carboxyl-terminal fragments containing the DNA-binding domain were highly phosphorylated, suggesting the presence of phosphorylation sites throughout the protein. Site-directed mutagenesis of wild type and deletion mutant AR at proline-directed consensus phosphorylation sites replaced Ser or Thr residues with Ala; wild type and mutant ARs were expressed in the presence of [32P]orthophosphate and isolated by immunoprecipitation using AR-specific antipeptide antibodies. Three proline-directed phosphorylation sites were identified: Ser 81 and 94 in the NH2-terminal region and Ser 650 in the hinge region. Expression of a series of NH2-terminal AR fragments provided evidence for additional sites in the NH2-terminal region. The effect of loss of each phosphorylation site on receptor function was determined by introducing the Ser to Ala mutations into full-length AR. Substituting Ser 81 and 94 with Ala had little effect on transcriptional activity when assayed by transient cotransfection. Substituting Ser 650 with Ala in the hinge region reduced transcriptional activity up to 30%. The results suggest at least three proline-directed phosphorylation sites in AR, one of which, serine 650, contributes to optimal gene activation by AR.