We developed a simplified method for quantitative measurement of trinitroglycerin in human plasma using hexane extraction and analysis by gas-liquid chromatography with electron-capture detection. This assay was linear from 0.5-60 ng/ml. Sensitivity and reproducibility were +/- 0.5 ng/ml. We used this assay to evaluate the pharmacodynamics of trinitroglycerin in 14 patients. Maximum plasma levels were similar with trinitroglycerin given by constant intravenous infusion (1.6 +/- 0.4 ng/ml (SEM)), transcutaneously (2.3 +/- 0.6 ng/ml), or sublingually (1.6 +/- 0.6 ng/ml). Despite similar levels and hemodynamic responses after intravenous trinitroglycerin, the dose range was wide (37.5-175 microg/min, n = 5), emphasizing the need to individualize therapy. In normal volunteers on no other drugs, the plasma level time course followed changes in heart rate better than blood pressure changes. Use of the trinitroglycerin assay may enhance optimization of trinitroglycerin therapy when administered by different methods.