Deep-sea sediments harbour a vast biosphere. Archaea—one of the three domains of life1—are prevalent in marine environments2, 3, 4, 5, and comprise a significant fraction of the biomass in marine sediments6. Archaeal membranes are well characterized, and are comprised of a glycerol backbone and a nonpolar isoprenoid chain. However, the ecology of sedimentary archaea remains elusive, because it is difficult to grow them in the laboratory. Here, we trace the fate of 13C-labelled glucose added to marine sediments in Sagami Bay, Japan, to determine the in situ mechanisms of membrane synthesis. Following the addition of labelled glucose to sediment samples collected in the region, we placed the cores on the sea floor and sampled them after 9 and 405 days. We found that the 13C was incorporated into the glycerol backbone of archaeal membranes; 13C was apparent after 9 days of incubation, but most pronounced after 405 days. However, the isoprenoid chain of the membranes remained unlabelled. On the basis of the differential uptake of 13C, we suggest that the glycerol unit is synthesized de novo, whereas the isoprenoid unit is synthesized from relic archaeal membranes and detritus, because of the prevalence of these compounds in marine sediments. We therefore suggest that some benthic archaea build their membranes by recycling sedimentary organic compounds