Riboflavin overproduction in the ascomycete Ashbya gossypii is limited by glycine, a precursor of purine biosynthesis, and therefore an indicator of glycine metabolism. Disruption of the SHM2 gene, encoding a serine hydroxymethyltransferase, resulted in a significant increase in riboflavin productivity. Determination of the enzyme's specific activity revealed a reduction from 3m-units/mg of protein to 0.5m-unit/mg protein. The remaining activity was due to an isoenzyme encoded by SHM1, which is probably mitochondrial. A hypothesis proposed to account for the enhanced riboflavin overproduction of SHM2-disrupted mutants was that the flux from glycine to serine was reduced, thus leading to an elevated supply with the riboflavin precursor glycine. Evidence for the correctness of that hypothesis was obtained from 13C-labelling experiments. When 500μM 99% [1-13C]threonine was fed, more than 50% of the label was detected in C-1 of glycine resulting from threonine aldolase activity. More than 30% labelling determined in C-1 of serine can be explained by serine synthesis via serine hydroxymethyltransferase. Knockout of SHM1 had no detectable effect on serine labelling, but disruption of SHM2 led to a decrease in serine (2—5%) and an increase in glycine (59—67%) labelling, indicating a changed carbon flux.