Anabaena variabilis ATCC 29413 and Anabaena PCC 7120 were grown in batch cultures with combined nitrogen source. The hox genes encoding the bidirectional hydrogenase were expressed at a relatively low level in the heterocyst-free filaments. Transcriptional regulation of the bidirectional hydrogenase in response to oxygen and light was found to be similar in the two closely related strains. Low oxygen tension caused c.100-fold induction of the hox genes. However, when the anaerobic cultures were incubated in darkness the hox transcript accumulation increased by one order of magnitude compared with the illuminated anaerobic cultures. In addition, darkness could also reversibly induce hox expression and was found to be a positive regulator of hox expression even in the presence of oxygen. These results imply a specific requirement for the bidirectional hydrogenase of Anabaena PCC 7120 and A. variabilis ATCC 29413 in darkness both in the presence and absence of oxygen.