Phosphorylation of the androgen receptor (AR) in human prostate tumor cells (LNCaP) is increased by androgens. The AR is expressed as two isoforms with apparent molecular masses of 110 and 112 kDa. Metabolic labeling experiments with [32P]orthophosphate revealed that only the 112 kDa isoform is radioactively labeled. Phosphoamino acid analysis revealed only phosphorylation on serine residues. Phosphotryptic peptide analysis of human AR protein by two-dimensional peptide mapping and by reverse-phase HPLC showed phosphorylation at multiple sites. Comparison of phosphopeptide maps of AR protein from cells incubated in the absence or presence of the synthetic androgen R1881 indicated that the ligand-stimulated phosphorylation is probably due to induction of phosphorylation at a new site rather than increased phosphorylation at an existing site. This result suggests that hormone-dependent AR phosphorylation might play a role in the signal transduction pathway of androgens.