Imaging and measurement of proliferation with computed tomography (CT) and positron emission tomography (PET) provide a noninvasive method for improved staging and monitoring of response to cancer treatment. We evaluated prospectively the proliferation marker 3'-deoxy-3'[18F] fluorothymidine (FLT) in the context of FLT-PET/CT for detection of early response, confirmation of posttreatment response, and prediction of relapse in dogs with non-Hodgkin's lymphoma. Nine dogs with non-Hodgkin's lymphoma who were scheduled to receive five cycles of an investigational cytotoxic chemotherapy agent were included. All dogs received baseline FLT-PET/CT imaging immediately before chemotherapy. Intent was to repeat imaging with FLT-PET/CT at various time points: group 1 (n = 4), 5 days after initiation of chemotherapy and 3 weeks following the last chemotherapy administration; group 2 (n = 5), before the fourth cycle of chemotherapy and 3 weeks following the last administration. Two dogs in group 2 did not undergo repeat PET/CT. Body mass standardized uptake values (SUV) for FLT were calculated for each dog. Eight dogs had initially increased FLT uptake (mean SUVmax = 9.8 [2.6-22.3]). Mean SUV decreased significantly for the seven dogs that underwent follow-up PET/CT following chemotherapy (mean SUVmax = 3.5 [1.1-7.9], P<0.016). Increased uptake preceded clinical and cytological evidence of relapse in two dogs. Ki-67 immunohistochemistry confirmed decreased proliferation corresponding to decreased SUV in three canine lymph node samples. FLT-PET/CT functional and anatomical imaging shows promise for the evaluation of response to cytotoxic chemotherapy in dogs with non-Hodgkin's lymphoma and for predicting relapse before standard clinical and clinicopathologic confirmation.