Serine/threonine phosphorylation of IRS-1 might inhibit insulin signaling, but the relevant phosphorylation sites are difficult to identify in cultured cells and to validate in isolated tissues. Recently, we discovered that recombinant NH₂-terminal Jun kinase phosphorylates IRS-1 at Ser³⁰⁷, which inhibits insulin-stimulated tyrosine phosphorylation of IRS-1. To monitor phosphorylation of Ser³⁰⁷ in various cell and tissue backgrounds, we prepared a phosphospecific polyclonal antibody designated αpSer³⁰⁷. This antibody revealed that TNF-α, IGF-1, or insulin stimulated phosphorylation of IRS-1 at Ser³⁰⁷ in 3T3-L1 preadipocytes and adipocytes. Insulin injected into mice or rats also stimulated phosphorylation of Ser³⁰⁷ on IRS-1 immunoprecipitated from muscle; moreover, Ser³⁰⁷ was phosphorylated in human muscle during the hyperinsulinemic euglycemic clamp. Experiments in 3T3-L1 preadipocytes and adipocytes revealed that insulin-stimulated phosphorylation of Ser³⁰⁷ was inhibited by LY294002 or wortmannin, whereas TNF-α–stimulated phosphorylation was inhibited by PD98059. Thus, distinct kinase pathways might converge at Ser³⁰⁷ to mediate feedback or heterologous inhibition of IRS-1 signaling to counterregulate the insulin response.