The rubredoxin expression level in Clostridium butyricum DSM 5431 grown in continuous culture was monitored using primer extension analyses of the rub gene and a specific enzymatic assay of the iron-sulfur protein. In this way, we showed that variations in rubredoxin content and in rub mRNA level were influenced by the pH of the culture and were directly dependent on the carbon flux. The maximum rubredoxin level reached 1227.3 pmol (mg of proteins)(-1) (i.e. 0.7% of the total protein content) under strictly anaerobic conditions when cells grew at pH 6.5 with an excess of glucose. In addition, primer extension analyses established that the control for all the variations observed operates at the level of gene transcription. Altogether, these results suggested a main function of rubredoxin in Clostridium butyricum independent of the protection against oxygen as has already been reported for Desulfovibrio gigas and Pyrococcus furiosus.