2-Chloro-N-(4-chloro-3-(pyridin-2-yl)-phenyl)-4-(methylsulfonyl)-benzamide (GDC-0449, vismodegib) is a potent and selective first-in-class small-molecule inhibitor of the Hedgehog signaling pathway and is currently in clinical development. In this study, we investigated the metabolic fate and disposition of GDC-0449 in rats and dogs after a single oral administration of [¹⁴C]GDC-0449. An average of 92.4 and 80.4% of the total administered radioactivity was recovered from urine and feces in rats and dogs, respectively. In both species, feces were the major route of excretion, representing 90.0 and 77.4% of the total dose in rats and dogs, respectively. At least 42.1 and 30.8% of the dose was absorbed in rats and dogs, respectively, based on the total excretion of radioactivity in bile and urine. GDC-0449 underwent extensive metabolism in rats and dogs with the major metabolic pathways being oxidation of the 4-chloro-3-(pyridin-2-yl)-phenyl moiety followed by phase II glucuronidation or sulfation. Three other metabolites resulting from an uncommon pyridine ring opening were found, mainly in feces, representing 1.7 to 17.7% of the dose in total in rats and dogs. In plasma, the total radioactivity was absorbed quickly in both rats and dogs, and unchanged GDC-0449 was the predominant circulating radioactive species in both species (>95% of total circulating radioactivity). Quantitative whole-body autoradiography in rats showed that the radioactivity was well distributed in the body, except for the central nervous system, and the majority of radioactivity was eliminated from most tissues by 144 h.