This work presents the characterization of an uptake hydrogenase from a marine filamentous nonheterocystous cyanobacterium, Lyngbya majuscula CCAP 1446/4. The structural genes encoding the uptake hydrogenase (hupSL) were isolated and characterized, and regulatory sequences were identified upstream of hupS. In silico analysis highlighted various sets of long repetitive sequences within the hupSL intergenic region and downstream of hupL. The transcriptional regulator that operates global nitrogen control in cyanobacteria (NtcA) was shown to bind to the promoter region, indicating its involvement in the transcriptional regulation of hupSL. Under N2-fixing conditions and a 12-h light/12-h dark regime, H2 uptake activity was shown to follow a daily pattern with a clear maximum towards the end of the dark period, preceded by an increase in the transcript levels initiated in the end of the light phase. Novel antibodies directed against HupL of Lyngbya majuscula CCAP 1446/4 were used to monitor the protein levels throughout the 24-h period. The results suggest that protein turnover occurs, with degradation taking place during the light phase and de novo synthesis occurring during the dark phase, coinciding with the pattern of H2 uptake. Taking into account our results and the established correlation between the uptake hydrogenase activity and N2 fixation in cyanobacteria, it seems probable that both processes are confined to the dark period in aerobically grown cells of Lyngbya majuscula CCAP 1446/4.