Abstract Adrenocortical carcinoma (ACC) is a rare disease with an overall poor but heterogeneous prognosis. This heterogeneity could reflect different mechanisms of tumor development. Gene expression profiling by transcriptome analysis led to ACC being divided into two groups of tumors with very different outcomes. Somatic inactivating mutations of the tumor suppressor gene TP53 and activating mutations of the proto-oncogene β-catenin (CTNNB1) are the most frequent mutations identified in ACC. This study investigates the correlation between p53 and β-catenin alterations and the molecular classification of ACC by transcriptome analysis of 51 adult sporadic ACCs. All TP53 and CTNNB1 mutations seemed to be mutually exclusive and were observed only in the poor-outcome ACC group. Most of the abnormal p53 and β-catenin immunostaining was also found in this group. Fifty-two percent of the poor-outcome ACC group had TP53 or CTNNB1 mutations and 60% had abnormal p53 or β-catenin immunostaining. Unsupervised clustering transcriptome analysis of this poor-outcome group revealed three different subgroups, two of them being associated with p53 or β-catenin alterations, respectively. Analysis of p53 and β-catenin target gene expressions in each cluster confirmed a profound and anticipated effect on tumor biology, with distinct profiles logically associated with the respective pathway alterations. The third group had no p53 or β-catenin alteration, suggesting other unidentified molecular defects. This study shows the important respective roles of p53 and β-catenin in ACC development, delineating subgroups of ACC with different tumorigenesis and outcomes. Cancer Res; 70(21); 8276–81. ©2010 AACR.