Abstract A high temperature NADPH recycling system was designed utilizing a thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from Bacillus stearothermophilus with glucose-6-sulfate (G6S) as the substrate. The thermostable alcohol dehydrogenase (tADH) from Thermoanaerobacter (formerly Thermoanaerobium) brockii was employed to catalyze the reduction of 2-butanone to 2-butanol at 55 °C. It was established that the B. stearothermophilus G6PDH is capable of utilizing G6S as the substrate ( K M=50 mM at 55 °C). NADPH recycling based on the G6S/tG6PDH system performed optimally with a 3:1 ratio of tG6PDH:tADH, and with 1 mM NADP. The system produced 75 mM 2-butanol and retained 60% of NADPH after 5 h at 55 °C. Under the same conditions, the system using glucose-6-phosphate (G6P), the natural substrate for tG6PDH, produced only 25 mM 2-butanol and retained negligible amount of the co-factor.