The slope environment of the Gulf of Mexico (GOM) supports dense communities of seep megafaunal invertebrates that rely on endosymbiotic bacteria for nutrition. Seep sediments also contain smaller macrofaunal invertebrates whose nutritional pathways are not well understood. Using stable-isotope analysis, we investigate the utilization of chemosynthetically fixed and methane-derived organic matter by macrofauna. Biological sampling was conducted in three lower-slope GOM seep environs: Green Canyon (GC852, 1428 m), Atwater Valley (AT340, 2230 m), and Alaminos Canyon (AC601, 2384 m). Infaunal delta¹³C and delta¹⁵N exhibited a broad range of values; most infauna appeared to be heterotrophic, although several taxa had very light delta¹⁵N and delta¹³C values, indicating possible reliance on chemoautotrophic symbioses. The lightest delta¹³C and delta¹⁵N values were observed in nematodes (delta¹³C=-54.6 + or - 0.1 per mil, delta¹⁵N=-6.1 + or - 0.2 per mil) and one gastropod (delta¹³C=-54.1 per mil, delta¹⁵N=-1.1 per mil) from Green Canyon. Mixing-model results indicated that sulfur-oxidizing Beggiatoa may be an important food source for seep infauna; the rate of utilization ranged from 60% to 100% at Green Canyon and Atwater Valley. The overall range in isotope values was similar across the three sites, suggesting that biogeochemical processes may be very similar in these geographically distinct areas.