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Insulin-like growth factor-I (IGF-I) stimulates the differentiation of preadipocytes, but the expression of IGF-I and IGF-binding proteins (IGFBPs) during the course of adipogenesis has not been investigated. Using two in vitro models, primary mouse preadipocytes and the 3T3-L1 preadipocyte cell line stimulated to differentiate with IGF-I, we studied IGF and IGFBP expression before and during differentiation. Primary preadipocyte cultures expressed IGF-I, IGFBP-2, and IGFBP-4 messenger RNAs (mRNAs), and conditioned medium (CM) contained IGFBP-3 [approximately 46,000 mol wt (M(r))], IGFBP-4 (24,000 M(r)), and a 30,000 M(r) IGFBP identified by immunoblot as IGFBP-2. During differentiation, an additional approximately 34,000 M(r) form of IGFBP-2 was predominant, but IGFBP-2 mRNA decreased, suggesting that a mechanism other than steady state mRNA levels is regulating protein abundance in CM. Like primary cultures, undifferentiated 3T3-L1 cells expressed IGFBP-4 mRNA, but insignificant levels of IGF-I and IGFBP-2 mRNAs. 3T3-L1 cell CM contained IGFBP-3 and IGFBP-4, and with the addition of IGF-I, a 30,000 M(r) IGFBP was also present. This IGFBP was not recognized by antiserum to IGFBP-1, -2, -4, -5, or -6. During differentiation of 3T3-L1 cells, an approximately 34,000 M(r) form of IGFBP-2 was also present in CM. In summary, primary cultures of mouse preadipocytes and 3T3-L1 cells express similar IGFBPs during IGF-I-stimulated adipogenesis. The presence of a larger isoform of IGFBP-2 in a differentiation-dependent manner and a potentially novel IGFBP in response to IGF-I suggests that these IGFBPs may be important in modulating IGF-I action in adipogenesis.