Nitric oxide (NO) is a small, gaseous, paramagnetic radical with a high affinity for interaction with ferrous hemoproteins such as soluble guanylate cyclase and hemoglobin. Interest in NO measurement increased exponentially with the discovery that NO or a related compound is the endothelium-derived relaxing factor (EDRF). In addition to being a potent endogenous vasodilator, NO has a role in inflammation, thrombosis, immunity, and neurotransmission. Measurement of NO is important as many of its effects (e.g., vasodilatation, inhibition of platelet aggregation) are similar to those of other substances produced by the endothelium, such as prostacyclin. NO is formed in small amounts in vivo and is rapidly destroyed by interaction with oxygen, making measurement difficult. A computerized search of the past five year's literature found NO measurements reported in fewer than 50 of 955 articles dealing with EDRF. Inhibitors of NO synthesis such as the arginine analogs or agents that inactivate NO, such as reduced hemoglobin, are commonly used as specific probes for NO, in vivo and in vitro; however, none of the NO inhibitors is completely specific. The most widely used assays use one of three strategies to detect NO: 1) NO is "trapped" by nitroso compounds, or reduced hemoglobin, forming a stable adduct that is detected by electron paramagnetic resonance (EPR) (detection threshold approximately 1 nmol); 2) NO oxidizes reduced hemoglobin to methemoglobin, which is detected by spectrophotometry (detection threshold approximately 1 nmol); 3) NO interacts with ozone producing light, "chemiluminescence" (detection threshold approximately 20 pmol). These assays can be performed to exclusively detect NO, or by adding acid and reducing agents to the sample, can measure NO and related oxides of nitrogen such as nitrite. Several new amperometric microelectrode assays offer the potential to measure smaller amounts of NO (10(-20) M), permitting NO measurement in intact issues and from single cells. This review describes the pharmacology and toxicology of NO and reviews the major techniques for measuring NO in biological models.