Three analogues of bovine parathyroid hormone (bPTH), [Tyr-34]bPTH-(1--34) amide, [Nle-8,Nle-18,Tyr-34]bPTH-(1--34)amide, and [Nle-8,Nle-18, o-NPS-Trp-23,Tyr-34]bPTH-(1--34)amide were synthesized by the solid-phase method. The synthetic peptides were found to be homogeneous in multiple analytical systems. These analogues represent the NH2-terminal one-third of the hormone, previously shown to contain all the structural requirements necessary for biological activity, but containing several structural modifications associated with enhancement and stabilization of biological activity. When tested in the in vitro renal adenylylcyclase assay, in which unsubstituted bPTH-(1--34) has a potency of 5400 MRC Units/mg, [Tyr-34]bPTH-(1--34)amide had a potency of 16,1000 MRC Units/mg, [Nle-8,Nle-18,Tyr-34]bPTH-(1--34)amide was 10,100 MRC Units/mg, and [Nle-8,Nle-18,o-NPS-Trp-23,Tyr-34]bPTH-(1--34)amide was 10,600 MRC Units/mg. The diverse structural features incorporated in these hormone analogues, resulting in a several-fold increase in biological activity in vitro, demonstrate additive effects on biological activity and should prove valuable in certain biological applications, as well as in the design of other parathyroid hormone analogues of enhanced potency.