Radiocarbon analyses of bulk carbon and individual organic compounds are presented for the hydrothermal environment of the Rebecca’s Roost vent in the southern trough of the Guaymas Basin hydrothermal field. The Δ 14 C values of CO 2 and CH 4 in the hottest hydrothermal fluids (317°C) are nearly “radiocarbon dead” (−944‰ and −923‰, respectively). In contrast, the Δ 14 C values of sediments and individual fatty acids (−418‰ to −227‰) obtained from a bacterial mat located south of the vent site are similar to values previously reported for hydrothermal petroleum in this environment and are more depleted in 14 C than overlying waters. Hydrothermal fluids moving through the sediments appear to supply 14 C of intermediate age to the bacteria. This carbon may take the form of, or may be supplied by processes similar to, the generation of hydrothermal petroleum. Although the bacterial mat visibly was dominated by Beggiatoa spp., such mats are known to include numerous other species. Individual compound data show that preaged carbon is being consumed by the integrated bacterial assemblage. Values of δ 13 C and Δ 14 C indicate that petroleum-derived carbon is incorporated directly into fresh bacterial biomass. Subsequently, some of this newly synthesized material also is consumed by heterotrophs, as eukaryotic sterols from the same sample also have 14 C-depleted values (Δ 14 C = −136‰ to −110‰). Therefore, the entire system may operate as a complex consortium to transform relict carbon back into biomass. Bacterial consumption of relict carbon occurs despite the ample supply of fresh carbon delivered from the productive, overlying water column.