This study investigates the proteolytic activation of latent transforming growth factor-β (TGFβ) from fibroblast-conditioned medium. TGFβ is typically produced in an inactive form by most cultured cells. The study used fibroblast-conditioned medium (NRK-49F and AKR-MCA cells) as a model to explore activation mechanisms. Active TGFβ was detected using radioreceptor assays, soft agar assays, antibody inhibition, and immunoprecipitation. Untreated conditioned medium showed little or no TGFβ activity. Extreme pH treatments (pH 1.5 or 12) significantly activated TGFβ, while mild acid treatment (pH 4.5) yielded only 20–30% of the activation achieved by pH 1.5. Proteases such as plasmin and cathepsin D were tested, and both generated 25-kD bands corresponding to active TGFβ. Plasmin treatment resulted in activity similar to mild acid treatment. Sequential treatments of conditioned medium with mild acid followed by plasmin or vice versa produced activation comparable to either treatment alone. These results suggest that conditioned medium contains at least two pools of latent TGFβ: one resistant to mild acid and/or plasmin, requiring strong acid or alkali for activation, and another activated by mild pH changes and/or plasmin. Activation of the latter form may occur through dissociation or proteolytic digestion from a precursor or hypothetical TGFβ-binding protein complex. The study also found that plasmin can activate a portion of latent TGFβ secreted by AKR-MCA and NRK-49F cells. Plasmin treatment resulted in activity similar to mild acid treatment, and plasmin-generated activity was inhibited by anti-TGFβ antibodies. The results suggest that TGFβ is relatively resistant to plasmin digestion. The study also compared acid and plasmin activation in soft agar assays, finding that both methods increased colony formation. Anti-TGFβ antibodies inhibited plasmin-generated activity, indicating that the activated TGFβ was immunoreactive. The study concludes that TGFβ may be activated by both extreme pH and proteolytic mechanisms, with plasmin being a potential physiological activator. The findings suggest that TGFβ activation is a critical step in its biological function, and that proteolytic activation may play a role in regulating cellular growth and extracellular matrix formation.This study investigates the proteolytic activation of latent transforming growth factor-β (TGFβ) from fibroblast-conditioned medium. TGFβ is typically produced in an inactive form by most cultured cells. The study used fibroblast-conditioned medium (NRK-49F and AKR-MCA cells) as a model to explore activation mechanisms. Active TGFβ was detected using radioreceptor assays, soft agar assays, antibody inhibition, and immunoprecipitation. Untreated conditioned medium showed little or no TGFβ activity. Extreme pH treatments (pH 1.5 or 12) significantly activated TGFβ, while mild acid treatment (pH 4.5) yielded only 20–30% of the activation achieved by pH 1.5. Proteases such as plasmin and cathepsin D were tested, and both generated 25-kD bands corresponding to active TGFβ. Plasmin treatment resulted in activity similar to mild acid treatment. Sequential treatments of conditioned medium with mild acid followed by plasmin or vice versa produced activation comparable to either treatment alone. These results suggest that conditioned medium contains at least two pools of latent TGFβ: one resistant to mild acid and/or plasmin, requiring strong acid or alkali for activation, and another activated by mild pH changes and/or plasmin. Activation of the latter form may occur through dissociation or proteolytic digestion from a precursor or hypothetical TGFβ-binding protein complex. The study also found that plasmin can activate a portion of latent TGFβ secreted by AKR-MCA and NRK-49F cells. Plasmin treatment resulted in activity similar to mild acid treatment, and plasmin-generated activity was inhibited by anti-TGFβ antibodies. The results suggest that TGFβ is relatively resistant to plasmin digestion. The study also compared acid and plasmin activation in soft agar assays, finding that both methods increased colony formation. Anti-TGFβ antibodies inhibited plasmin-generated activity, indicating that the activated TGFβ was immunoreactive. The study concludes that TGFβ may be activated by both extreme pH and proteolytic mechanisms, with plasmin being a potential physiological activator. The findings suggest that TGFβ activation is a critical step in its biological function, and that proteolytic activation may play a role in regulating cellular growth and extracellular matrix formation.