The marine filamentous nonheterocystous nitrogen-fixing cyanobacterium Lyngbya aestuarii (F. K. Mert.) Liebman ex Gomont CCY 9616 was grown under diazotrophic and nondiazotrophic conditions and under an alternating 16:8 light:dark (L:D) regime. Nitrogenase activity appeared just before the onset of the dark period, reaching its maximum 1–2 h in the dark, subsequently decreasing to zero at the beginning of the following light period. Nitrogenase activity was only detected at low levels of O2 (5%) and when the culture was grown in the absence of combined nitrogen. Quantitative reverse transcriptase–PCR (RT-PCR) analysis of one of the structural genes encoding nitrogenase, nifK, showed that the highest levels of transcription preceded the maximum activity of nitrogenase by 2–4 h. nifK transcription was not completely abolished during the remaining time of the 24 h cycle. Even in the presence of nitrate, when nitrogenase activity was undetectable, nifK was still transcribed. The H2-uptake activity seemed to follow the nitrogenase, but the transcription of hupL (gene encoding the large subunit of uptake hydrogenase) preceded the nifK transcription. However, H2-uptake and hupL transcription occurred throughout the 24 h cycle as well as under nondiazotrophic conditions, albeit at much lower levels. The hoxH transcript levels (a structural gene coding for the bidirectional hydrogenase) were similar under diazotrophic or nondiazotrophic conditions but slightly higher during the dark period. All three enzymes investigated are involved in H2 metabolism. It is concluded that the uptake hydrogenase is mainly responsible for H2 uptake. Nevertheless, uptake hydrogenase and nitrogenase do not seem to be coregulated.