Rabbit antisera were raised against synthetic phosphopeptides corresponding to defined or putative sites of insulin receptor serine/threonine phosphorylation (Ser-1305, Ser-1327, Thr-1348). All of these antibodies bound specifically to the immunogenic phosphopeptide but not to the non-phosphorylated form of the peptide or to other phosphopeptides, in a microtitre plate competition enzyme-linked immunosorbent assay. Anti-PS1327 antibody reacted well with native insulin receptor prepared from phorbol ester-treated transfected CHO.T cells, but showed little reaction with receptor from untreated cells. Anti-PT1348 antibody in crude form reacted substantially with receptor from both phorbol 12-myristate 13-acetate-treated and untreated cells, but displayed specificity for phosphoreceptor after adsorption to remove antibodies reactive with dephosphopeptide. The ability to discriminate between receptor from cells treated with or without phorbol ester was retained when these antibodies were used to probe denatured receptor on Western blots. Thus anti-PS1327 and anti-PT1348 react with insulin receptor in a site-specific and phosphorylation-state-dependent manner. Anti-PT1348, but not anti-PS1327, also showed increased reactivity with receptor prepared from insulin-treated cells. The third antibody, anti-PS1305, did not react with intact insulin receptor under any conditions. It is concluded that serine 1327 is a major, previously unrecognized, site of phorbol ester-induced receptor phosphorylation, and that anti-phosphopeptide antibodies will be valuable reagents with which to examine the serine/threonine phosphorylation state of receptor extracted from tissues.