AbstractLarge, mesopelagic teleost fishes have a potentially keystone position in the ecology of the pelagic water column, yet remain relatively unstudied when compared with large, commercially important, epipelagic fishes. Here, the ecological roles of four, large, vertically migrating teleosts were examined. Stomach content analyses were performed on 48 oilfish (Ruvettus pretiosis), 35 escolar (Lepidocybium flavobrunneum), 32 snake mackerel (Gempylus serpens) and seven lancetfish (Alepisaurus spp.) collected from pelagic longline gear in the Western North Atlantic Ocean from 2007 to 2010. Of these specimens, stable carbon and nitrogen isotope analyses were also performed on white dorsal muscle tissue from 33 oilfish, 16 escolar, 27 snake mackerel and seven lancetfish. Based on literature length‐at‐maturity values, all escolar, snake mackerel and lancetfish specimens were mature, while 13 of the 33 oilfish were juveniles. Crustaceans, annelids, salps, cephalopods and teleosts were present in the stomachs and were presumed to be prey items. A Kruskal–Wallis test showed the four species to be isotopically segregated in both δ13C and δ15N. Escolar were the most depleted in δ13C, followed by adult oilfish, juvenile oilfish and lancetfish, with snake mackerel the most enriched. The depletion in δ13C of adult oilfish and escolar may have been attributable to high C/N values, which were strongly correlated with length in oilfish, weakly correlated with length in escolar and moderately correlated with length in snake mackerel and lancetfish. The high C/N was likely due to the high lipid concentration of these fishes. Other factors that may have contributed to the depletion in δ13C may include spawning or a change in carbon source within the ecosystem. Large escolar occupied the highest trophic level (δ15N = 10.20), followed by snake mackerel (δ15N = 9.66), adult oilfish (δ15N = 9.32), lancetfish (δ15N = 9.05) and juvenile oilfish (δ15N = 7.83). A marked change in oilfish δ13C and C/N at 30–35 cm fork length coincided with a presumed length‐at‐maturity.