Germ-line protein kinase A (PKA) regulatory-subunit type-Iα (RIα; PRKAR1A)-inactivating mutations and loss-of-heterozygosity (LOH) of its 17q22–24 locus have been found in Cushing syndrome (CS) caused by primary pigmented nodular adrenocortical disease (PPNAD). We examined whether somatic 17q22–24, PRKAR1A, or PKA changes are present in 44 sporadic adrenocortical tumors (29 adenomas and 15 cancers); 26 of these tumors were responsible for CS. A probe containing the PRKAR1A gene–mapped by fluorescent in situ hybridization to 17q22–24–and corresponding microsatellite markers were used to study allelic losses; PRKAR1A was sequenced in all samples. 17q22–24 losses were seen in 23 and 53% of adenomas and cancers, respectively. In three tumors, somatic, PRKAR1A-inactivating mutations were identified: (a) a nonsense mutation in exon 6 (A751G); (b) a splicing mutation (9IVS-1G/A); and (c) a transition (1050T>C) followed by a 22-bp deletion, also in exon 9; all predicted premature RIα protein terminations. Quantitative message and protein studies showed RIα down-regulation in tumors with genetic changes; their cortisol secretion pattern was similar to that of PPNAD, and they had higher PKA activity by enzymatic studies. We conclude that somatic allelic losses of the 17q22–24 region, PRKAR1A-inactivating mutations or down-regulation, and corresponding PKA activity changes are present in at least some sporadic adrenocortical tumors, especially those with a PPNAD-like clinical presentation of CS.