To evaluate the use of urinary biomarkers to assess exposure of cats to environmental tobacco smoke (ETS). 61 healthy client-owned cats (19 from households in which smoking was reported and 42 from households in which there was no smoking). 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. 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). 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.