No studies have investigated the hormonal regulation of IGF-binding protein-2 (IGFBP-2) secretion and mRNA expression in myoblasts. In this study, cells of the C2C12 mouse myoblast cell line were used to examine the effects of various agents on the hormonal regulation of IGFBP-2. Conditioned medium (CM) was collected and cells were harvested at 2, 4, 6, 15 and 24 h after exposure to treatment media containing porcine insulin (pINS) or recombinant human IGF-I (rhIGF-I), and at 6, 15 and 24 h after exposure to treatment media containing dexamethasone (DEX) or prostaglandin E2 (PGE2). Relative abundance of a single 1.8 kb IGFBP-2 mRNA transcript was determined by Northern analysis using total cellular RNA and a labeled cDNA specific for rat IGFBP-2. IGFBP-2 was detected in CM by probing Western blots with 125I-IGF-I (ligand blot analysis). We have previously shown by immunoblot analysis that the predominant 32 000 Mr protein on ligand blots is IGFBP-2. Treatment with 10-9 or 10-6 M pINS led to a rapid reduction (P<0.01) in relative IGFBP-2 mRNA abundance and protein secretion relative to controls. Treatment with 7 × 10-10 10 or 7 × 10-9 M (5 or 50 ng/ml) rhIGF-I increased IGFBP-2 mRNA abundance and protein secretion (P<0.01). Cultures treated with 10-8 M DEX exhibited significantly increased (P<0.001) IGFBP-2 mRNA and protein. IGFBP-2 secretion was not affected by 10-6 M PGE2 but mRNA levels were higher than controls at 24 h (P<0.01). These findings suggest that multiple factors, including growth factors and metabolic hormones, are involved in regulating IGFBP-2 expression in C2C12 myoblasts.