The ability to induce an autocatalytic reaction by mixing P2, H2O2 and a reducing agent has been investigated in a Tris buffer at pH 8.5. As a reductant, we used the same enzyme/reducing substrate as previously, i.e. the diaphorase/NADH couple. The diaphorase (DI) from Bacillus stearothermophilus (EC 1.6.99.-) is a thermostable flavin mononucleotide-dependent oxidoreductase that efficiently catalyzes the obligatory two-electron reduction of naphthoquinones into naphthohydroquinones in the pH range 7 to 10. It is also insensitive to O2 and its NADH substrate is quite stable in slightly basic aerated buffers (showing almost no cross-reactivity with dissolved dioxygen unless either O2 or NADH is activated ). The time course of the autocatalytic reaction was monitored by UVvisible spectrophotometry at  = 340 nm, a wavelength characteristic of the transformation of NADH into NAD + . A spectrophotometric microplate reader for high-throughput kinetic analysis was used to simultaneously monitor a set of different reaction mixtures (200 µL working solution per microwell). During the whole reaction, the microplate was left open to the atmosphere so as to operate under an approximately constant O2 concentration.