At the onset of glucose-limited continuous cultures, riboflavin production in recombinant Bacillus subtilis declines significantly within 3 generations. This phenomenon was specific to riboflavin production and was not correlated with any other physiological parameter. Physiological analyses excluded genetic degeneration or co-metabolism of previously generated overflow metabolites as possible causes for the riboflavin transients. By developing a novel method for (13)C-based metabolic flux analysis under non-steady-state conditions, we showed that the pentose precursors of riboflavin were exclusively synthesized via the non-oxidative pentose-phosphate (PP) pathway as long as riboflavin production was high. The complete redirection of carbon flux to the oxidative branch of the PP pathway was achieved at unaltered PP pathway gene expression and correlated with the declining riboflavin production. With the possible exception of a slight down-regulation of the purine biosynthesis pathway, genome-wide expression analysis indicated that transcriptional regulation was not responsible for the production decline.