Green nonsulfur-like bacteria (GNSLB) in hot spring microbial mats are thought to be mainly photoheterotrophic, using cyanobacterial metabolites as carbon sources. However, the stable carbon isotopic composition of typical Chloroflexus and Roseiflexus lipids suggests photoautotrophic metabolism of GNSLB. One possible explanation for this apparent discrepancy might be that GNSLB fix inorganic carbon only during certain times of the day. In order to study temporal variability in carbon metabolism by GNSLB, labeling experiments with [13C]bicarbonate, [14C]bicarbonate, and [13C]acetate were performed during different times of the day. [14C]bicarbonate labeling indicated that during the morning, incorporation of label was light dependent and that both cyanobacteria and GNSLB were involved in bicarbonate uptake. 13C-labeling experiments indicated that during the morning, GNSLB incorporated labeled bicarbonate at least to the same degree as cyanobacteria. The incorporation of [13C]bicarbonate into specific lipids could be stimulated by the addition of sulfide or hydrogen, which both were present in the morning photic zone. The results suggest that GNSLB have the potential for photoautotrophic metabolism during low-light periods. In high-light periods, inorganic carbon was incorporated primarily into Cyanobacteria-specific lipids. The results of a pulse-labeling experiment were consistent with overnight transfer of label to GNSLB, which could be interrupted by the addition of unlabeled acetate and glycolate. In addition, we observed direct incorporation of [13C]acetate into GNSLB lipids in the morning. This suggests that GNSLB also have a potential for photoheterotrophy in situ.