In an investigation of the spatial characteristics of Laurentian Great Lakes food webs, we examined the trophic relationship between benthic amphipods (Diporeia) and plankton in Lake Superior. We analyzed the carbon and nitrogen stable isotope ratios of Diporeia and plankton at stations in water column depths of 4300 m. Neither δ15N nor δ13C of plankton from the upper 50 m of the water column varied significantly with station depth. Diporeia isotope ratios exhibited depth-specific patterns reflecting changes in food sources and food web relationships with plankton. Diporeia was 13C enriched at station depths of <40 m, reflecting increased dietary importance of benthic algae. There was a systematic increase in Diporeia δ15N with depth, which appeared to result from a combination of dietary shifts in the nearshore and decompositional changes in Diporeia's principal food, sedimented plankton, in deep habitats. Diporeia δ13C and δ15N together described changes in food web isotope baseline with depth. They also discriminated three depth strata representing photic, mid-depth, and profundal zones. These findings have implications for our understanding of Great Lakes food webs and analyses of trophic position within them, the ecology of zoobenthos and plankton communities, and sampling designs for large lakes.