Three mammalian isoforms of transforming growth factor-β (TGFβ) are known, TGFβ1, 2, and 3, that have non-overlapping functions during development. However, their specific roles in cancers such as prostate cancer are less clear. Here we show that primary cultures of prostatic epithelial cells preferentially produce and activate the latent TGFβ2 isoform. Paired cultures of normal and malignant prostate cells from prostate cancer patients produced predominantly the TGFβ2 isoform, with 30- to 70-fold less TGFβ1. By mono-Q ion exchange chromatography, three major peaks of latent TGFβ2 activity were observed corresponding to the known small latent TGFβ2 complex, the known large latent TGFβ2 complex and a novel eluting peak of latent TGFβ2. Although prostate cells are known to activate latent TGFβ, the mechanism for activation is currently unclear. We investigated whether prostate specific antigen (PSA), a serine protease used as a clinical marker for prostate cancer, could play a role in the activation of latent TGFβ. Unlike plasmin, a known activator of both latent TGFβ1 and 2, PSA specifically activated the recombinant small latent form of TGFβ2, but not TGFβ1. Prostate epithelial cells, therefore, preferentially produce the TGFβ2 isoform and PSA, a protease produced by the prostate, specifically targets the activation of this TGFβ isoform. PSA-mediated activation of latent TGFβ2 may be an important mechanism for autocrine TGFβ regulation in the prostate and may potentially contribute to the formation of osteoblastic lesions in bone metastatic prostate cancer. © 2004 Wiley-Liss, Inc.