trans-3-Hydroxycotinine is the major urinary metabolite of nicotine in man and can serve as an important biomarker of tobacco smoke exposure. A sensitive ELISA test for trans-3-hydroxycotinine was developed with an I-50 for this nicotine biomarker of between 1.0–3.0 ng/ml. This ELISA test has about 10 fold less affinity for cotinine and 1000-fold less affinity for nicotine and other nicotine metabolites. No matrix effects were detectable in human saliva and relatively small matrix effects (I-50 for trans-3-hydroxycotinine, about 25 ng/ml) in urine was observed. The assay readily detected levels of apparent trans-3-hydroxycotinine in urine samples from smoke-exposed mice and rats. This ELISA is therefore a sensitive test for the determination of trans-3-hydroxycotinine in plasma, saliva, and urine samples from humans and animals, and can be used to monitor exposure to tobacco smoke or nicotine.