Intracellular NADPH/NADP(+) ratio in cells grown on various production media with different carbon and nitrogen sources had a positive correlation with the thymidine production. To improve thymidine production in a previously engineered E. coli strain, NAD(+) kinase was overexpressed in it resulting in the NADPH/NADP(+) ratio shifting from 0.184 to 0.267. The [NADH + NADP(+)]/[NAD(+) + NADPH] ratio was, however, not significantly altered. In jar fermentation, 740 mg thymidine l(-1) was produced in parental strain, while 940 mg l(-1) of thymidine was produced in NAD(+) kinase-expressing strain.