Insulin stimulates the autophosphorylation of the insulin receptor (IR) on multiple tyrosine residues in three domains: tyrosines 1146, 1150 and 1151 in the 1150 domain, 1316 and 1322 in the C-terminal domain and tyrosines in at least one other domain (possibly 953, 960 or 972). The ability of calcineurin to dephosphorylate the autophosphorylated IR was examined, characterised and compared with the dephosphorylation of the autophosphorylated epidermal growth factor (EGF) receptor by calcineurin. Calcineurin dephosphorylated the ³²P-labelled EGF receptor > 3 times faster than the ³²P-labelled IR; both dephosphorylations had comparable dependencies on metal ions and calmodulin. Native phospho-tyrosyl protein phosphatase (PTPP) activity in cell extracts was also characterised. With rat liver, heart or brain the majority (71%) of PTPP activity against ³²P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell. The ³²P-labelled EGF receptor was dephosphorylated > 2 times the rate of the ³²P-labelled IR by both the soluble and particulate fractions. Properties of particulate and soluble PTPP activity against ³²P-labelled insulin and EGF receptors were similar and are reported. Ni²⁺ (1 mM) was found to be a potent new inhibitor of PTPP activity [99.5% ± 6% (mean ± SEM, 30 observations) inhibition of insulin and EGF receptor PTPP activity in particulate and soluble fractions from rat liver, heart and brain]. In contrast Ni²⁺ markedly activated calcineurin. This and other methods were used to show that in cell extracts of rat liver, heart and brain PTPP(s) other than calcineurin were the major phosphotyrosyl insulin and EGF receptor phosphatases. The synthetic 1142-1153 peptide of the IR was phosphorylated on tyrosine by the IR and found to be a potent substrate for dephosphorylation by rat liver particulate and soluble PTPP(s). Apparent Km values were approximately 5 μM. V_max values (nmol ³²Pi removed/min/mg protein) were 0.62 (particulate) and 0.2 (soluble). This corresponded to 80% of the PTPP activity being membrane associated. Properties of rat liver particulate and soluble PTPP activities with the ^32P-labelled 1142-1153 peptide as substrate were similar to those found with the ^32P-labelled receptors as substrates. The sequence of dephosphorylation of IR autophosphorylation sites was examained and found to be similar with either particulate or soluble PTPP preparations from rat liver. The triphosphorylated 1150 domain was dephosphorylated 3-10 fold faster than either the di- or monophosphorylated 1150 domain or the phosphorylation sites in other domains. The major sequence of dephosphorylation of the 1150 domain was tyrosine 1150 or 1151, follwed by tyrosine 1146, yielding the 1150 domain monophosphorylated mainly (> 80%) at tyrosine 1150 or 1151. Dephosphorylation of the C-terminal domain was ordered; tyrosine 1316 then tyrosine 1322. As triphosphorylation of the IR in the 1150 domain appears important in activating the tyrosine kinase to phosphorylate other proteins, the exquisite sensitivity of this species to dephosphorylation may be important in terminating or regulating IR tyrosine kinase action and insulin signalling.