Objective-To evaluate splenic mast cell tumors (MCT) of cats for activating mutations in the protooncogene c-kit. Sample Population-10 formalin-fixed, paraffinembedded splenic MCT from cats in the pathology database of the Veterinary Medical Teaching Hospital at the University of California, Davis. Procedure-Genomic DNA was isolated from tumor specimens, and the polymerase chain reaction (PCR) procedure was performed for exons 11, 12, and 17. The PCR products were analyzed by use of agarose gel electrophoresis and then directly sequenced. Results-We did not identify mutations in the juxtamembrane domain (encoded by exons 11 and 12) or catalytic domain (encoded by exon 17) of c-kit in any of the splenic MCT specimens. Conclusions and Clinical Relevance-Although mutations in the proto-oncogene c-kitoccur frequently in naturally developing MCT in dogs and aggressive mastocytosis in humans, the data reported here documented that dysregulation of Kit function through activating mutations is unlikely in splenic MCT of cats. Therapeutic strategies aimed at inhibiting Kit signaling (ie, kinase inhibitors such as imatinib [STI571]) may not be of benefit for the treatment of this disease in cats.