Somatically acquired, activating mutations of GNAS, the gene encoding the stimulatory G-protein Gsalpha subunit, have been identified in kidney, thyroid, pituitary, leydig cell, adrenocortical and, more recently, in colorectal tumours, suggesting that mutations such as R201C may be oncogenic in these tissues. To study the role of GNAS in intestinal tumourigenesis, we placed GNAS R201C under the control of the A33-antigen promoter (Gpa33), which is almost exclusively expressed in the intestines. The GNAS R201C mutation has been shown to result in the constitutive activation of Gsalpha and adenylate cyclase and to lead to the autonomous synthesis of cyclic adenosine monophosphate (cAMP). Gpa33(tm1(GnasR201C)Wtsi/+) mice showed significantly elevated cAMP levels and a compensatory upregulation of cAMP-specific phosphodiesterases in the intestinal epithelium. GNAS R201C alone was not sufficient to induce tumourigenesis by 12 months, but there was a significant increase in adenoma formation when Gpa33(tm1(GnasR201C)Wtsi/+) mice were bred onto an Apc(Min/+) background. GNAS R201C expression was associated with elevated expression of Wnt and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) pathway target genes, increased phosphorylation of ERK1/2 MAPK and increased immunostaining for the proliferation marker Ki67. Furthermore, the effects of GNAS R201C on the Wnt pathway were additive to the inactivation of Apc. Our data strongly suggest that activating mutations of GNAS cooperate with inactivation of APC and are likely to contribute to colorectal tumourigenesis.