Liver-type fatty acid-binding protein (L-FABP) is expressed by several tissues, plays a role in fatty acid metabolism, and has antioxidant effects. Its renal expression is upregulated by stress. Urinary L-FABP (uL-FABP) is a promising kidney biomarker in people for detection of early acute and chronic kidney disease (CKD) and as a marker for progression in patients with glomerulonephritis. However, data on canine uL-FABP are currently limited. This prospective study was designed to examine canine tissue expression of L-FABP and to validate an ELISA to quantify uL-FABP in older dogs with or without early signs of CKD. Tissues of 4 recently euthanized dogs and 117 urine samples of 73 client-owned older dogs undergoing health screening were evaluated in the study. Immunohistochemistry was performed on kidney and liver tissues. Analytical validation of a commercially available ELISA for measurement of L-FABP in canine urine was performed (limit of detection, imprecision, specificity). The ELISA was used to measure L-FABP in stored urine samples from a cohort of older dogs. Dogs were found to express L-FABP, mostly in proximal tubular epithelial cells and in the periportal hepatocytes of the liver. Assay validation revealed poor sensitivity and imprecision for measurement of canine uL-FABP. Of the 117 urine samples analyzed, 98 were below the limit of detection (LOD
7.30 ng/mL) and a further 5 were below the limit of quantification (LOQ
16.20 ng/mL). The proximal tubules of dog kidneys express L-FABP, but the value of uL-FABP as tubular marker in older dogs warrants further study.