We cloned a novel splicing variant for nuclear coactivator p120(alpha), designated as p120beta and studied its function and expression in several human prostate diseases. Transfection assays demonstrated that p120beta functions as a strong coactivator for androgen receptor (AR), but weakly for other nuclear receptors. GST-pull down assay showed that a glutamine-rich region of the p120 bound to the ligand-binding domain of AR. Interestingly, p120beta mRNAs were expressed predominantly in the normal prostate, androgen-responsive prostate cancers and an androgen-sensitive prostate cancer cell line, LNCaP, but weakly in recurrent cancers and the androgen-insensitive prostate cancer cell lines PC3 and DU145. Furthermore, knockdown of p120alpha by siRNA abolished coactivator activity on thyroid hormone receptors (TR) and PPARgamma, but did not affect that of ARs in PC3 cells. In addition, competitive assay with other nuclear receptors demonstrated that TR and PPARgamma did not inhibit p120beta-induced stimulation. These findings suggested that while p120alpha was essential for ligand-dependent stimulation of TRs and PPARgamma, p120beta acted as a coactivating protein predominantly for AR.