We have developed a novel method for the continuous assay of protein disulfide oxidoreductase activity using as substrate bovine pancreas insulin in which both N-terminal amino groups are chemically modified with fluorescein isothiocyanate. The reduction of intercatenary disulfide bonds of di-fluoresceinthiocarbamyl-insulin with dithiothreitol initially lowers but subsequently enhances the emission intensity. In this biphasic kinetics, the rate of increase is sensitive enough for the estimation of Escherichia coli thioredoxin concentrations from 5 nM (0.06 microgram/ml) to 500 nM (6 micrograms/ml). Neither changes of pH over a range of 6.2 to 8.4 nor neutral salts (K+, Mg2+, and Ca2+) at concentrations lower than 100 mM affect this simple reaction system. Moreover, the fluorometric method is functional for measuring the reductive capacity of Brassica napus protein disulfide isomerase. Hence, a highly reproducible and accurate one-state assay for protein disulfide oxidoreductase activity not only greatly improves the sensitivity compared to the commonly used turbidimetric assay but also represents a reliable alternative to assays based on accessory enzymes or radiolabeled substrates.