Objective - To evaluate the use of urinary biomarkers to assess exposure of cats to environmental tobacco smoke (ETS). Animals - 61 healthy client-owned cats (19 from households in which smoking was reported and 42 from households in which there was no smoking). Procedures - Urine samples were obtained from each cat and assayed for total nicotine (nicotine plus nicotine glucuronide) and total cotinine (cotinine plus cotinine glucuronide) content by use of gas chromatography-mass spectrometry. In addition, total urinary content of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, was measured by use of gas chromatography with nitrosamine-selective detection. Results - Cats from households in which smoking was reported had significantly higher concentrations of total nicotine (70.4 ng/mL), total cotinine (8.53 ng/mL), and total NNAL (0.0562 pmol/mL) in urine, compared with concentrations for cats that lived in households in which there was no smoking (4.89 ng/mL, 0.74 ng/mL, and 0.0182 pmol/mL, respectively). Conclusions and clinical relevance - Analysis of these data provided biochemical evidence of exposure to ETS and uptake of tobacco-specific carcinogens by cats that live in households with smokers. Biomarkers could facilitate investigation of the health effects of ETS in cats and other species.