IRS-1 plays an essential role in mediating the insulin signals that trigger mitogenesis, lipid synthesis and uncoupling protein-1 gene expression in mouse brown adipocytes. Expression of IRS-3 is restricted mainly to white adipose tissue; expression of this IRS protein is virtually absent in brown adipocytes. We have tested the capacity of IRS-3 to mediate insulin actions in IRS-1-deficient brown adipocytes. Thus, we expressed exogenous IRS-3 in immortalized IRS-1-/- brown adipocytes at a level comparable to that of endogenous IRS-3 in white adipose tissue. Under these conditions, IRS-3 signaling in response to insulin was observed, as revealed by tyrosine phosphorylation of IRS-3 and the activation of PI-3 kinase associated with this recombinant protein. However, although insulin promoted the association of Grb-2 with recombinant IRS-3 in IRS-1-/-cells, the exogenous expression of this IRS family member failed to activate p42/44 MAPK and mitogenesis in brown adipocytes lacking IRS-1. Downstream of PI 3-kinase, IRS-3 expression restored insulin-induced Akt phosphorylation, which is impaired by the lack of IRS-1 signaling. Whereas the generation of IRS-3 signals enhanced SREBP1c/ADD-1 and FAS mRNA and protein expression, activation of this pathway was unable to reconstitute C/EBPa and UCP-1 transactivation and gene expression in response to insulin. Similar results were obtained following IGF-I stimulation. In brown adipocytes expressing the IRS-3F4 mutant, the association of the p85a regulatory subunit via SH2 binding was lost but insulin nevertheless induced PI 3-kinase activity and Akt phosphorylation in a wortmannin-dependent manner. In contrast, activation of IRS-3F4 signaling failed to restore the induction of SREBP1c/ADD-1 and FAS gene expression in IRS1-deficient brown adipocytes. These studies demonstrate that recombinant IRS-3 may reconstitute some, but not all, of the signals required for insulin action in brown adipocytes. Thus, our data further implicate a unique role for IRS-1 in triggering insulin action in brown adipocytes.