The amplification approach centered on the cycling of two reversibly interconvertible chemical species sequentially participating in two different enzyme-catalyzed reactions (enzymatic amplification by substrate cycling) has been implemented on-line into a continuous-flow/stopped-flow/continuous-flow operation. The implementation is illustrated with the determination of L-lactate in a dual enzyme reactor containing immobilized lactate oxidase (LOD, EC 1.1.3.x) to catalyze the oxidation of L-lactate by dissolved oxygen. The immobilized LOD was affixed to a rotating disk in the lower part of the flowthrough cell. Immobilized lactate dehydrogenase (EC 1.1.1.27), affixed to the top part of the cell regenerates L-lactate with the mediation of (\beta) NADH as the hydrogen donor. The substrate cycling permits the generation of (\mathrm{H}{2} \mathrm{O}{2}) beyond the stoichiometric limitation, and this is detected at a stationary Ptring electrode located at the bottom part of the cell. The stationary Pt-ring electrode is positioned concentrically to the rotating disk containing the immobilized LOD. The resulting amplified response permits, in a simple manner, achievement of detection limits as low as (0.3 \mathrm{fmol} \cdot \mathrm{liter}^{-1}) and allows the processing of 30 samples per hour. 1994 Academic Press. Inc.