We previously showed that when added to fresh medium, insulin-like growth factor (IGF)-II stimulates glycogen synthesis in cultured 18-day-old fetal rat hepatocytes. In the present study, we investigated the influence of 24-h culture-conditioned medium on IGF-II- and insulin-induced glycogenesis. The stimulatory effect of IGF-II (2.9-fold) on [14C]glucose incorporation into glycogen over 3 h was dose dependently inhibited by conditioned medium, whereas that of insulin (3.2-fold) was unaffected. Western ligand and immunoblot analysis of the conditioned media revealed IGF binding protein (IGFBP)-1, IGFBP-2, and IGFBP-4, with a predominance of IGFBP-1. IGFBP-3 was not detected. Preincubation of conditioned medium with IGF-II for 4 h at 4 C restored the glycogenic effect of newly added IGF-II. Preincubation of fresh medium with recombinant IGFBP-1 or IGFBP-3 in the presence of IGF-II suppressed IGF-II stimulation of glycogen synthesis. IGFBPs alone had no effect on glycogenesis. Des(1-6)IGF-II and R6IGF-II, structural analogs of IGF-II that have weak affinity for the IGFBPs, elicited maximal stimulation, near that of IGF-II (2.8-fold and 3.1-fold vs. 3.0-fold for IGF-II), whether tested in fresh or conditioned medium. The inhibitory effect of conditioned medium on IGF-II-induced glycogenesis is therefore mediated by the IGFBPs via sequestration of IGF-II. This suggests that the IGFBPs, particularly IGFBP-1, produced by fetal rat hepatocytes in culture may play a role in regulating glycogenesis.