This study investigates the activation of latent transforming growth factor-β (TGFβ) from fibroblast-conditioned medium. TGFβ is typically produced by cultured cells in an inactive form, and the mechanisms of its activation are not well understood. The authors used fibroblastic (NRK-49F and AKR-MCA) cell-conditioned medium as a model to study potential activation mechanisms. They found that active TGFβ was detected in the medium after treatment with extreme pH (1.5 or 12), while mild acid treatment (pH 4.5) resulted in only 20-30% activation. Proteases, particularly plasmin and cathepsin D, were tested for their ability to activate TGFβ. Plasmin treatment of the medium produced activity similar to that of mild acid treatment, and this activity was inhibited by anti-TGFβ antibodies. Sequential treatments with mild acid followed by plasmin or plasmin followed by mild acid gave comparable activation levels to either treatment alone. These findings suggest that the conditioned medium contains at least two pools of latent TGFβ: one resistant to mild acid and/or plasmin, requiring strong acid or alkali treatment for activation, and another activated by mild pH changes and/or plasmin. The activation of the latter form may involve dissociation or proteolytic digestion from a precursor molecule or a hypothetical TGFβ-binding protein complex. The study also discusses the potential physiological significance of TGFβ activation by plasmin and the possibility of a negative feedback mechanism involving TGFβ-regulated plasminogen activator activity.This study investigates the activation of latent transforming growth factor-β (TGFβ) from fibroblast-conditioned medium. TGFβ is typically produced by cultured cells in an inactive form, and the mechanisms of its activation are not well understood. The authors used fibroblastic (NRK-49F and AKR-MCA) cell-conditioned medium as a model to study potential activation mechanisms. They found that active TGFβ was detected in the medium after treatment with extreme pH (1.5 or 12), while mild acid treatment (pH 4.5) resulted in only 20-30% activation. Proteases, particularly plasmin and cathepsin D, were tested for their ability to activate TGFβ. Plasmin treatment of the medium produced activity similar to that of mild acid treatment, and this activity was inhibited by anti-TGFβ antibodies. Sequential treatments with mild acid followed by plasmin or plasmin followed by mild acid gave comparable activation levels to either treatment alone. These findings suggest that the conditioned medium contains at least two pools of latent TGFβ: one resistant to mild acid and/or plasmin, requiring strong acid or alkali treatment for activation, and another activated by mild pH changes and/or plasmin. The activation of the latter form may involve dissociation or proteolytic digestion from a precursor molecule or a hypothetical TGFβ-binding protein complex. The study also discusses the potential physiological significance of TGFβ activation by plasmin and the possibility of a negative feedback mechanism involving TGFβ-regulated plasminogen activator activity.