Plasma concentrations of 1,2- and 1,3-glyceryl dinitrates were measured in each of four healthy volunteers who received intravenous infusions (10, 20, and 40 micrograms/min), topical ointment (20 mg/200 cm2), and oral solution (6.5 mg) doses of nitroglycerin and from two subjects who received sublingual (0.4 mg) nitroglycerin. The ratio of 1,2-glyceryl dinitrate to 1,3-glyceryl dinitrate (dinitrate ratio) was determined for each subject after each dose and was found to vary with route of administration. Dinitrate ratios were 7.36, 4.60, 3.86, and 1.99 for intravenous, sublingual, topical, and oral doses, respectively. Nonspecific metabolism of nitroglycerin would result in twice as much of the 1,2-dinitrate as the 1,3-dinitrate (i.e., a dinitrate ratio of 2.0, such as that produced after oral administration). A high ratio (e.g., after intravenous administration) indicates that the metabolism was more specific. These results indicate that metabolite formation depends on route of administration, implying different metabolic specificity of enzymes in the gut, liver, skin, sublingual mucosa, and blood vessels.