The unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 (Cyanothece 51142) is able to grow aerobically under nitrogen-fixing conditions with alternating light-dark cycles or continuous illumination. This study investigated the effects of carbon and nitrogen sources on Cyanothece 51142 metabolism via (13)C-assisted metabolite analysis and biochemical measurements. Under continuous light (50 mumol photons m(-2) s(-1)) and nitrogen-fixing conditions, we found that glycerol addition promoted aerobic biomass growth (by twofold) and nitrogenase-dependent hydrogen production [up to 25 mumol H(2) (mg chlorophyll)( -1) h(-1)], but strongly reduced phototrophic CO(2) utilization. Under nitrogen-sufficient conditions, Cyanothece 51142 was able to metabolize glycerol photoheterotrophically, and the activity of light-dependent reactions (e.g. oxygen evolution) was not significantly reduced. In contrast, Synechocystis sp. PCC 6803 showed apparent mixotrophic metabolism under similar growth conditions. Isotopomer analysis also detected that Cyanothece 51142 was able to fix CO(2) via anaplerotic pathways, and to take up glucose and pyruvate for mixotrophic biomass synthesis.