AbstractFrom medium conditioned by 3T3 cells, we had previously purified an inhibitory factor of Mr 45 kDa which we termed IDF45 (inhibitory diffusible factor). The protein was able to 100% inhibit stimulation induced in CEF by 1% calf serum and to reversibly prevent cell growth. We then demonstrated that IDF45 was an IGF-binding protein. Our results suggested that IDF45 was a bifunctional molecule able to bind IGF and to inhibit DNA synthesis stimulated by this hormone, but also to inhibit stimulation of DNA synthesis induced by another growth factor in serum. Indeed, its N terminal amino acid sequence has great homology with that of IGFBP-3 and IDF45 is now proposed to be named IGFBP-3 (mouse IGF binding protein). Present results show that Ha-ras transfected 3T3 cells (EJ cells), like 3T3 cells, secrete a mIGFBP-3 molecule. In addition, transfected cells secrete a doublet of an IGF-binding protein (IGFBP-28) of Mr 28 kDa which is not secreted by untransformed 3T3 cells. IGFBP-28 has been purified and characterized in this work. Various results suggest that IGFBP-28 is not a degradation product of mIGFBP-3. Its N terminal amino acid sequence was different from that of mIGFBP-3. IGFBP-28 inhibited DNA synthesis stimulated by IGF-I, but much more IGFBP-28 protein than mIGFBP-3 was required to prevent this stimulation. In agreement with this result, IGFBP-28 has low affinity for IGF-I. In contrast, IGFBP-28 has high affinity for IGF-II. Like mIGFBP-3, IGFBP-28 was able to inhibit the stimulation induced by serum in CEF and to reversibly prevent growth, though with a specific activity lower than that of mIGFBP-3. It has also the capacity to inhibit stimulation of DNA synthesis induced by high molecular weight serum proteins depleted in IGF-I and II.In conclusion we have shown that transformation of 3T3 cells with Ha-ras induced the synthesis of a new IGF binding protein in medium conditioned by normal 3T3 cells. Our results suggest that IGFBP-28 like mIGFBP-3 is a bifunctional protein able to inhibit stimulation induced by IGF and by serum proteins different from IGFs.