The continental slope of the Gulf of Mexico supports dense aggregations of tubeworms and mussels that have symbiotic chemoautotrophic bacteria. Associated with these communities are numerous heterotrophic fauna and free-living bacteria. Here we examine the stable C, N, and S isotope compositions of fauna from two chemoautotrophic communities to identify isotope ranges of chemoautotrophic primary production and determine the usage of that primary production by heterotrophic invertebrates. The range in isotope values of the chemoautotrophic production is different between sites. A brine seep (GC233) dominated by mussels symbiotic with methanotrophic bacteria has ¹³C and ¹⁵N depleted nutrient sources (−50 to −65‰ and −9 to −12‰, respectively), indicating methanotrophy using biogenic methane and suggesting ammonium as the dominant nitrogen source. However, those same sources were ³⁴S-enriched (6‰–11‰), as indicated by resident heterotrophs (Munidopsis sp., Methanoaricia dendrobranchiata Blake 2000, Alvinocaris stactophila Williams 1988, Phascolosoma turnerae Rice 1985), indicating that thiotrophy was a minor chemosynthetic method at the site. A site dominated by tube worms and mussels (GC234) has two isotopically distinct sources of carbon, one between −24 and −30‰ and another of approximately −40‰, as indicated by the resident heterotrophs. Resident heterotrophs at GC 234 had δ¹⁵N and δ³⁴S values from 1‰ to 5‰ and −10‰ to 6‰, respectively. These isotope values suggest a mix of thiotrophy and methanotrophy (largely from thiotrophic sources) at the site. We estimate that hagfish (Eptatretus sp.) captured approximately 2 km from the communities derived at least 10% of their carbon from chemoautotrophic sources because of low δ¹³C values. In contrast, giant isopods (Bathynomus giganteus Milne Edwards, 1879), captured with the hagfish show negligible incorporation of chemosynthetic production.