Abstract—Lipid biomarkers and ¹³C fractionation patterns were used to understand the dynamics of carbon cycling during microbial metabolisms in different environments of travertine precipitation (called facies) at Spring AT-1 on Angel Terrace in the Mammoth Hot Springs complex of Yellowstone National Park, USA. Microbial mats that encrust travertine deposits were collected for analyses of lipid biomarkers and carbon isotopes along the continuous drainage outflow system of Spring AT-1. The spring water exhibits a continuous temperature drop from 71°C in the vent at top to 24°C in the distal slope at bottom. Phospholipid fatty acids (PLFA) and glycolipid fatty acids (GLFA) exhibit distinctly different compositions in each of the facies, which are consistent with partitioning of the bacterial 16S rRNA gene sequences in the Spring AT-1 travertine facies (Fouke et al., 2003). The δ¹³C composition of total biomass within the microbial mats decreases from -16.1‰ in the vent to -23.5‰ in the distal slope. However, lower values occur in the pond (-26.0‰) and the proximal slope (-28.0‰) between the vent and the distal slope. Isotopic compositions of PLFA and GLFA have variations similar to those of total biomass. The average δ¹³C values of PLFA are -12.4 ± 5.2‰ (n = 10 individual fatty acids, same below) in the vent, -33.0 ± 3.1‰ (n = 11) in the pond, -33.7 ± 3.8‰ (n = 16) in the proximal slope, and -22.4 ± 3.4‰ (n = 10) in the distal slope; the average δ¹³C values of GLFA are -19.6 ± 3.0‰ (n = 3) in the vent, -30.4 ± 4.7‰ (n = 8) in the pond, -36.9 ± 2.8‰ (n = 12) in the proximal slope, and -27.9 ± 3.1‰ (n = 13) in the distal slope. In particular, fatty acids in the vent are enriched in ¹³C relative to the total biomass, which is consistent with the notion that the biosynthetic pathways of the extant microbial community in the vent may be dominated by Aquificales using the reversed tricarboxylic acid cycle. Fractionations between fatty acids and total biomass in the pond, the proximal slope and the distal slope suggest the involvement of other biosynthetic pathways for CO₂ fixation by extant microbial populations. The results indicate that lipid biomarkers provide valuable information on the changing diversity and activity of microbial communities in different depositional environments. Carbon-isotope fractionations, on the other hand, can provide insight into the operating biosynthetic pathways associated with different organisms in the changing environment. This integrated approach may serve as a powerful tool for identifying functional metabolism within a community and identify shifts in microbial community structure in modern hot-spring systems.