A latent β-TGF activity is spontaneously released into serum-free culture medium by chicken embryo fibroblasts. Anchorage-independent growth activity measured on NRK-49F indicator cells, of this latent β-TGF can be revealed by four different treatments: acidification, alkalinisation, exposure to urea, and heating to 100° C for 3 minutes. This lact activating treatment indicates that latent β-TGF activation in vitro is non-enzymatic. Active β-TGF exists in a low molecular weight form 16 Kd (apparent) in 1M acetic acid, which elutes on reverse phase (FPLC) between 33–35% acetonitrile. Under neutral conditions only a high molecular weight form excluded on Biogel P60 is observed. This form is poorly active on NRK-49F for anchorage independent growth but can be fully activated by prior acidification. Rechromatography of the latent β-TGF-containing fractions under acidic conditions converts the high molecular weight form to an apparent 16 Kd active form. We suggest that the high molecular weight form may correspond to a complex of a β-TGF associated with a carrier or binding protein.