A potent competitive inhibitor of PTH-stimulated biological responses in vitro, [Nle8,Nle18,Tyr34] bovine PTH (bPTH)-(3-34)amide, was evaluated in vivo in dogs. These studies confirm observations in vitro, suggesting that positions 1 and 2 of the peptide are critical to its biological activity. However, unlike the results from studies in vitro, this PTH analog is a weak agonist with effects on parathyroid target tissues that produce hypercalcemia and phosphaturia and increase urinary cAMP excretion. Assessed by these three parameters of hormonal action in vivo, the estimated potency of this analog is less than 1% of that of the intact hormone. In addition, PTH-induced biological responses were not inhibited by relatively large doses of the bPTH-(3-34) analog. These results emphasize the need for a systemic, integrated approach, combining chemical with biological studies, to design effective inhibitors of hormonal action in vivo. Although the rationale for introducing particular modifications into the peptide structure is most frequently based on bioassays performed in vitro, the success of the strategy chosen must rely, ultimately, upon the demonstration of specific biological properties in vivo.