Adrenocortical tumor (ACT) is a rare, heterogeneous malignancy whose pathogenesis is unclear. The oncoprotein PTHrP, found in many common tumors, can regulate their growth in an autocrine/paracrine fashion through the receptor PTH-R1. Little is known about the role of PTHrP in ACT. We monitored the synthesis of PTHrP and PTH-R1 in a series of 25 ACT: 12 adrenocortical carcinoma (ACC), 13 adrenocortical adenoma (ACA), and investigated the effects of PTHrP (134) on H295R cells derived from adrenocortical carcinoma. PTH-R1 mRNA and proteins were detected by RT-PCR and western blotting in all the ACT samples and in H295R cells. There was no significant difference between their concentrations in the ACT. PTHrP mRNA was assayed by quantitative real-time PCR. It was detected in 90% of ACC, and in 10% of ACA. There was a positive correlation between the usual pronostic factors (tumors size and weight, MacFarlane stages, Weiss Score), and steroid hormone production. Tissue-specific PTHrP protein processing was shown by western blotting. Immunohistochemical staining revealed numerous, dense foci of PTHrP-containing cells in ACC, but few positive cells in ACA or normal tissue. PTHrP stimulated the growth of H295R cells, while specific anti-PTHrP antibody and PTHrP-R1 antagonist both enhanced their apoptosis. PTHrP did indeed activate the cell cycle in H295R cells and released the G1/S checkpoint, resulting in a redistribution of cells in the S and G2/M phases. PTHrP activated both adenylate cyclase/protein kinase A and the intracellular calcium /protein kinase C pathways via PTHrP-R1. The active synthesis of PTHrP is linked to a poor prognosis in ACC, where it may act as an autocrine/paracrinre factor in tumor growth and malignancy.