We have developed a single-stage assay for heparin, using reagents modified from the two-stage Dade Protopath heparin synthetic substrate assay. The single-stage assay involves simultaneous mixing of a plasma sample, an antithrombin III source, alpha-thrombin, and the alpha-thrombin fluorogenic substrate. The synthetic substrate, antithrombin III, and heparin-antithrombin III complex compete for the alpha-thrombin active site. The alpha-thrombin is inactivated by the heparin-antithrombin complex while substrate is being hydrolyzed, so that total product formation decreases with heparin concentration. Day-to-day CV was 9.3% at a heparin concentration of 246 USP units/L. Comparison of results of the single-stage heparin assay with those of a two-stage esterolytic assay yielded the linear regression equation: esterolytic = 0.834 (single-stage)--7 USP units/L (r = 0.94, n = 47). Bilirubin interfered with the single-stage assay, resulting in an apparent increase in sample heparin concentration. The single-stage heparin assay can be automated for centrifugal analyzers capable of double-reagent addition and fluorometric detection, substantially decreasing reagent requirements and therefore costs.