On the otherwise low-biomass seafloor of the Gulf of Mexico (GoM) continental slope, natural oil and gas seeps are oases of local primary production that support lush animal communities. Hundreds of seep communities have been documented on the continental slope, and nutrition derived from seeps could be an important link in the overall GoM food web. Here, we present a uniquely large and cohesive data set of δ(13)C, δ(15)N, and δ(34)S compositions of the vestimentiferan tubeworms Escarpia laminata and Lamellibrachia sp. 1, which dominate biomass at GoM seeps and provide habitat for hundreds of other species. Our sampling design encompassed an entire region of the GoM lower slope, allowing us for the first time to assess spatial variability in isotope compositions and to robustly address long-standing hypotheses about how vestimentiferans acquire and cycle nutrients over their long lifespan (200+ years). Tissue δ(13)C values provided strong evidence that larger adult vestimentiferans use their buried roots to take up dissolved inorganic carbon from sediment pore water, while very small individuals use their plume to take up carbon dioxide from the seawater. δ(34)S values were extremely variable among individuals of the same species within one location (<1 m(2) area), indicating high variability in the inorganic sulfur pools on a very small spatial scale. This finding supports the hypothesis that vestimentiferans use their roots to cycle sulfate and sulfide between their symbionts and free-living consortia of sulfate-reducing archaea in the sediment. Finally, consistent differences in δ(15)N between two cooccurring vestimentiferan species provided the first strong evidence for partitioning of inorganic resources, which has significant implications for the ecology and evolution of this taxonomic group.