We have purified peptides with PTH-like bioactivity from a rat Leydig cell tumor (H-500) and a human squamous cell carcinoma, both associated with a syndrome of humor-induced hypercalcemia. Tumor extracts were shown to be active in an in vitro renal cytochemical bioassay and in an in vitro osteosarcoma cell (UMR 108) adenylate cyclase assay; activity in both assays could be reduced by the PTH antagonist [norleucine-8,18,tyrosine-34]bovine PTH-(3-34)-amide. Partially purified extracts of both tumors and of rat tumor-conditioned culture medium were active in vivo in thyroparathyroidectomized rats in preventing hypocalcemia and increasing fractional phosphorus excretion and cAMP excretion. Ion exchange chromatography demonstrated that active peptides were basic in character. Employing reverse phase HPLC and gel permeation HPLC, active peptides of approximately 9,000 and 9,500 daltons were purified from extracts of the human and rat tumors, respectively, which had similar but not identical compositions. Two additional bioactive peptides were detected in rat tumor extract, and the more active had a mol wt of approximately 28,000. The results demonstrate that peptides that mimic PTH in a variety of in vivo and in vitro bioassays can be extracted from malignancies associated with hypercalcemia, that multiple molecular species may be detected in tumors that demonstrate PTH-like activity, and that at least one of these peptides may be similar in two tumors of highly divergent cell and species origin.