Bottom hypoxia has reemerged as a prominent feature of Lake Erie's central basin during late summer. Similar to coastal and marine systems, the influence of hypoxia on pelagic organisms remains largely enigmatic in Lake Erie. During 2005, we used a plankton survey system (a sensor package consisting of an optical plankton counter, fluorometer, dissolved oxygen sensor, light sensor, and conductivity–temperature–depth sensor), coupled with a fish hydroacoustics system, to explore how the distribution of phytoplankton (chlorophyll), mesozooplankton, and fish varied vertically and horizontally in relation to oxygen concentrations. To do so, we conducted surveys of the entire water column on a continuous basis during mild (August) and severe (September) hypoxia. Our surveys included two sampling designs: 1) basin-wide transects sampled during day and night to define broad-scale patterns of spatial overlap among pelagic organisms; and 2) shorter (5 km) transects sampled every 4 h over a 24-h period to explore how diel vertical migration and hypoxia interact to affect time-specific spatial overlap among fishes, mesozooplankton, and phytoplankton. Our findings indicated that fish avoided regions of the hypolimnion with dissolved oxygen concentrations