Insulin-like growth factor II (IGF-II) stimulates the proliferation and differentiation of rat myoblasts. Previous studies suggest that these response are mediated by the IGF-I receptor, but the IGF-II/mannose 6-phosphate receptor was recently implicated in differentiation of mouse myoblasts. L6A1 myoblasts synthesize IGF-binding protein-4 (IGFBP-4), IGFBP-5, and IGFBP-6, which modulate IGF action. We studied the roles of IGF receptors and IGFBPs in L6A1 myoblast proliferation and differentiation by comparing the effects of IGF-II and a number of IGF-II mutants with decreased affinities for IGF receptors and/or IGFBPs. IGF-II induced concentration-dependent proliferation with a maximum increase of 47%; half-maximal proliferation was seen with approximately 50 ng/ml. [Arg54, Arg55]IGF-II bound to the IGF-I receptor with slightly lower affinity than IGF-II, did not bind to the IGF-II/mannose 6-phosphate receptor, and bound to IGFBPs secreted by myoblasts with approximately 16-fold decreased affinity. It induced proliferation with equal potency to IGF-II. [Leu27]IGF-II, which did not bind to the IGF-I receptor but bound to the IGF-II/mannose 6-phosphate receptor and IGFBPs with slightly lower affinity than IGF-II, had a markedly impaired proliferative effect, inducing proliferation only at high concentrations. [Thr48, Ser49, Ile50]IGF-II, which bound to the IGF-I receptor with slightly lower affinity than IGF-II but did not substantially bind to the IGF-II/mannose 6-phosphate receptor or IGFBPs, induced proliferation with approximately 5-fold greater potency than IGF-II. The order of potency in inducing myoblast differentiation was the same, although there was less difference in the relative potencies of IGF-II and mutants. Coincubation of recombinant human (rh) IGFBP-6 in molar excess with IGF-II inhibited myoblast proliferation and differentiation. rhIGFBP-6 was slightly less potent did not inhibit proliferation or proliferation or differentiation induced by [Thr48,Ser49,Ile50]IGF-II. These results suggest that 1) IGF-II-induced proliferation and differentiation of L6A1 myoblasts are predominantly mediated by the IGF-I receptor; 2) the IGF-II/mannose 6-phosphate receptor is not required for these actions of IGF-II; 3) nevertheless, the IGF-II/mannose 6-phosphate receptor may be capable of mediating these actions; and 4) IGFBPs secreted by myoblasts inhibit IGF actions.