LUTEINIZING hormone stimulates testicular Leydig cells to produce testosterone by binding to a receptor that activates the G protein G_s and adenylyl cyclase. Testotoxicosis is a form of precocious puberty in which the Leydig cells secrete testosterone in the absence of luteinizing hormone, often due to constitutive activation of the luteinizing hormone receptor and (indirectly) G_s (refs 1–4). Here we study two unrelated boys suffering from a paradoxical combination of testotoxicosis and pseudohypoparathyroidism type la (PHP-Ia)⁵, a condition marked by resistance to hormones acting through cyclic AMP (parathyroid hormone and thyroid-stimulat-ing hormone) as well as a 50% decrease in erythrocyte Gs activity (the remaining 50% is due to the normal G_s allele)^5,6. In both patients, a mutation in the gene encoding the G_s α-subunit replaced alanine at position 366 with serine⁵. We show that this α_s-A366S mutation constitutively activates adenylyl cyclase in vitro, causing hormone-independent cAMP accumulation when expressed in cul-tured cells, and accounting for the testotoxicosis phenotype (as cAMP stimulates testosterone secretion). Although as-A366S is quite stable at testis temperature, it is rapidly degraded at 37 °C, explaining the PHP-Ia phenotype caused by loss of G_s activity. In vitro experiments indicate that accelerated release of GDP causes both the constitutive activity and the thermolability of α_s-A366S.