The effects of bST injection and dietary protein level on blood hormone and metabolite concentrations were examined in four mature Holstein cows in a double crossover design. Cows were assigned at d 5 to 9 postpartum to receive daily injections of either a control (saline) solution or 20.6 mg of bST. Four 3-wk periods were used during which one cow from each group was fed a medium protein diet (17.1% CP), and the other received a high protein diet (23.6% CP). Injections of bST or control solutions began on d 0 of the second period. Intakes of DM were not influenced by dietary protein or bST injection. Milk yield tended to increase with increased CP level but was not affected by bST injection. Based on the rate and extent of decline in milk production after cessation of bST injection, the cows assigned to bST had lower milk production potential than control cows. Thus, the effect of bST injection apparently was to enhance milk yield to levels similar to those of controls. There were no significant CP level or bST injection effects on glucose, FFA, somatostatin, or somatotropin concentrations. Glucagon concentrations were higher in bST-treated cows. Concentrations of insulin-like growth factor-I were increased with increased CP level and also with bST injection. Significant effects of days on bST were observed for insulin, insulin-like growth factor-I, glucose, and FFA. Cows given bST injections and producing equal amounts of milk as control cows did not show major physiological differences in hormones and metabolites with the exception of insulin-like growth factor-I.