The structure of the biologically active mutant PTHrP[Ala-26](1-34)amide in 10% trifluoroethanol was studied by two-dimensional proton NMR spectroscopy. Complete assignments of all backbone and side chain hydrogens were made with the aid of totally correlated and nuclear Overhauser effect spectroscopy. The NMR data were utilized in the distance geometry algorithm (DIANA) and the resulting family of structures further refined using dynamic simulated annealing (X-PLOR). The major structural features include two segments of alpha-helix extending from Glu-4 to Lys-13 and from Phe-21/Phe-22 to Ala-34, with a turn from Gln-16 to Arg-19 and a hinge around Ser-14/Ile-15. A close resemblance to the structure of PTH(1-34)amide in water was noted. A comparison of the structural features common to PTH and PTHrP in different solvents was made which enabled the key structural features likely to be involved in PTH receptor binding to be identified.