The effects of insulin, using a 4 d hyperinsulinemic-euglycemic clamp, on plasma concentrations of hormone, metabolites, and binding proteins were evaluated in four Holstein dairy cows during wk 4 and 17 of lactation. Insulin was infused at 1 microg/kg/hr for 96 hr during the clamp period. Compared with the pre-clamp period, plasma insulin concentrations increased 7-fold and 4-fold during the clamp periods in early and mid-lactation, respectively. The total amount of glucose infused was higher (P < 0.05) during the clamp in early lactation. The clamp decreased plasma concentrations of non-esterified fatty acids (P < 0.001) during early lactation while differences in mid-lactation were minor. The clamp also decreased plasma concentration of beta-hydroxybutyrate (P < 0.001), plasma urea nitrogen (P < 0.001), and true protein (P < 0.01) although the patterns of decline differed between early and mid-lactation. Growth hormone (GH) concentrations decreased (P < 0.001) and insulin-like growth factor-1 (IGF-1) increased (P < 0.01) during the clamp period suggesting a direct effect of insulin on the un-coupling of the GH/IGF-1 axis. Levels of IGF binding protein-2 (IGFBP-2) decreased (P < 0.01) during the clamp period. The relative proportion of IGFBP-2 decreased (P < 0.001) and that of IGFBP-3 increased (P < 0.001) during the clamp period. There were no interactions between the clamp period and stage of lactation on GH, IGF-1, or IGFBPs. Overall, most plasma variables measured were affected in the same way during the two clamps, but the pattern of change often varied with stage of lactation.