Previous deletion studies established that the 25-34 region of PTH is important for receptor binding. We used oligonucleotide-directed mutagenesis to generate 47 different mutations in this region of human (h) PTH-(1-84) and evaluated cAMP-stimulating activity in ROS 17/2.8 cells. The hydrophobic residues Leu24 and Leu28 stood out as mutationally intolerant sites, while neighboring polar residues were comparatively tolerant. A series of synthetic PTH analogs was designed to test these residues further. The affinity of [Tyr34]hPTH-(1-34)NH2 for ROS 17/2.8 cells [dissociation constant (Kd), approximately 5 nM)] was dramatically reduced by the substitution of either Leu24 or Leu28 with Glu (Kd, approximately 20,000 and 8,000 nM, respectively). The Val31-->Glu substitution also sharply reduced affinity (Kd, approximately 200 nM). In contrast, the nearby charge-reversing change of Asp30-->Lys had no effect on binding affinity (Kd, approximately 5 nM). Similar effects were observed in the opposum kidney cell line. The binding of [Tyr34]hPTH-(15-34)NH2 to ROS 17/2.8 and opposum kidney cells (Kd, approximately 10 microM) was abolished by Glu substitutions at position 24, 28, or 31; the Lys30 change was without effect. These results suggest that the adverse effects of the Glu substitutions on receptor binding are not due purely to the disruption of tertiary interactions with the 1-14 region. Circular dichroism spectroscopy indicated that the substitutions do not affect local helical structure. The data suggest that Leu24, Leu28, and Val31 contribute important receptor-binding interactions and are consistent with the hypothesis that an amphipathic alpha-helix in the carboxy-terminal region of PTH-(1-34) is involved in receptor binding.