A high recovery protocol was devised for the isolation of a 25Kd protein in a pure form from bovine serum using successive affinity and gel permeation chromatography techniques. Structural characterization of the resulting isolate shows that it is a Mr 25Kd polypeptide consisting of two chains held together with disulphide bonds. 25Kd is an acid- and heat-stable molecule, requiring the disulphide bonds for full activity. The biological activity of 25Kd is in a latent form that requires acidification, alkinisation or exposure to urea for activation. When tested on different cell lines for mitogenicity, 25Kd shows a 7.2- and a 3-fold increase over basal at 50ng/ml with CH23 and SV3T3 fibroblasts respectively. However, it shows a considerable inhibition (up to 90% at 10ng/ml with 3T3 cultures. Active 25Kd stimulates colony formation on soft agar of CH23, 3T3 and SV3T3 which requires the presence of 5ng/ml epidermal growth factor (EGF). The concentration of purified 25Kd required to elicit a half-maximal response for formation of colonies is 0.1-5ng/ml when assayed in the presence of 5ng/ml EGF. Both anti- 25Kd antibodies and synthetic pentapeptide Gly-Arg-Gly-Asp-Ser inhibit colony-stimulating activity of 25Kd. The finding that 25Kd is not a fibronectin fragment provides more supporting evidence that 25Kd is a serum growth factor. The structural properties, chromatographic behaviour, immunological and biological activities of 25Kd suggest a close relationship to transforming growth factor type beta (TGF-B).