Hydrothermal fluids circulate through extensive areas of the upper oceanic crust. Most hydrothermal circulation occurs on ridge flanks(1,2), where low-temperature fluids flow through porous basalts. These fluids contain variable levels of dissolved organic carbon, but the source and composition of this carbon are uncertain. Here, we report Delta C-14 and delta C-13 measurements of dissolved organic carbon in ridge-flank and on-axis hydrothermal fluids sampled from the Juan de Fuca Ridge. Dissolved organic carbon from two independent ridge-flank sites was characterized by low delta C-13 and Delta C-14 values. The delta C-13 values ranged from -26 to -35 parts per thousand, and were consistent with a chemoautotrophic origin. The C-14 ages of the dissolved organic carbon ranged from 11,800 to 14,400 years before present, revealing that the carbon was around three times older than dissolved organics in the deep ocean. The Delta C-14 values of the ridge-flank dissolved organic matter also corresponded closely to those of dissolved inorganic carbon in the same fluid samples. Taken together, the data suggest that chemosynthetic crustal microbial communities synthesize dissolved organic carbon from inorganic carbon in ridge-flank fluids. We suggest that ridge-flank circulation may support an indigenous biosphere extensive enough to export substantial fixed carbon, with distinct isotopic and probably compositional character, to the overlying ocean.