We have previously reported that scorbutic and fasted guinea pig sera contain an insulin-like growth factor-I (IGF-I)-reversible inhibitor of collagen, proteoglycan, and DNA synthesis in cultured cells. Here we report that IGF-binding protein (IGFBP) activity is increased in serum containing the inhibitor [125I]IGF-I or -II bound to these sera was eluted in the 30- to 50-kDa region of an S200 gel column. [125I]IGF-I affinity cross-linking analysis revealed that a 38-kDa cross-linked species increased markedly in fasted and scorbutic sera, with a lesser increase in a 34-kDa species, while scorbutic sera also yielded a 44-kDa species. Gel filtration of unlabeled sera showed a 10-fold increase in the activity of two proteins in the 30- to 50-kDa region from the experimental sera. Their activity correlated with their ability to inhibit binding of [125I]IGF-I to its cellular receptor, suggesting that they have the potential to inhibit IGF-I-dependent functions. Ligand blotting showed that 29 and 35-kDa IGFBPs were almost undetectable in normal serum, but were dramatically induced by scurvy and fasting, so that they accounted for close to 40% of the total circulating BPs. Total IGFBP-3 in the experimental sera was increased about 30%, while there was little effect of scurvy or fasting on the level of BP-3 activity isolated by acid extraction of the high mol wt region of the S200 column. An IGF-I analog with normal affinity for the 30- to 50-kDa BPs from fasted and scorbutic sera, but with reduced affinity for the cell receptor, was equivalent to IGF-I in reversing the inhibition of collagen synthesis by scorbutic guinea pig serum in human fibroblasts. Thus, reversal of inhibition appears to require initial saturation of IGFBPs. The overall results suggest that two circulating IGFBPs with unoccupied binding sites are induced in vitamin C-deficient or fasted guinea pigs and may be responsible for inhibition of IGF-I-dependent functions by sera from these animals.