Squamous carcinomas are the most common cause of humoral hypercalcemia of malignancy (HHM) in humans. To develop an animal model of this syndrome, CD-1 female mice were painted with dimethylbenzanthracene, which produced cutaneous squamous carcinomas in the majority of those painted. Greater than 90% of tumor-bearing mice developed a syndrome of hypercalcemia, hypophosphatemia, hypercalciuria, elevated plasma 1,25-dihydroxyvitamin D, normal immunoreactive PTH, elevated urinary cAMP, and accelerated bone resorption compared to control mice. Tumor excision reversed the hypercalcemia and hypophosphatemia, and autopsies revealed no evidence of skeletal or other metastases. Dietary calcium restriction did not affect the hypercalcemia in tumor-bearing mice. Extracts of tumor tissue contained potent bioactivity paralleling that of bovine (b) PTH in a PTH-sensitive canine renal cortical adenylate cyclase assay. The activity was trypsin sensitive and partially inhibitable by Nle, Tyr bPTH amide. The activity coeluted with chymotrypsinogen (mol wt, 25,700) on Sephacryl S-200 chromatography, well ahead of bPTH. This is the first description of an animal squamous carcinoma that produces HHM. With the exception of elevated plasma 1,25-dihydroxyvitamin D levels, the syndrome precisely mimics that seen in human HHM. The presence of a biologically active protein larger than PTH in tumor extracts, similar to that extracted from human tumors, suggests a common mode of pathogenesis. This model should be useful in further studying the pathophysiology of HHM.