Oligotrophication of Lake Ontario has led to increased water clarity and an increased proportion of zooplankton residing in the metalimnion during the day, which may affect the utilization of different depth regions for planktivorous fish. We investigated day and night distributions of fish using hydroacoustics and suspended vertical gillnets during the summer of 2013 when a deep chlorophyll layer (DCL) was established. We related fish distributions to concurrent measures of temperature and prey (zooplankton) density. Alewife dominated in vertical gill net catches, indicating that most acoustic targets were alewife. Alewife schooled during the day in the bottom of the mixed layer, and at dusk alewife schools broke up and fish moved towards the surface. We hypothesize this movement followed migrating zooplankton to allow feeding at night; alewife sampled from vertical gillnets fed on cyclopoid copepods and cladocerans, prey groups that migrate into the epilimnion at night. Some alewife remained at the bottom of the mixed layer at night and these fish ate deep-water calanoid copepods such as Limnocalanus. Vertical distributions were best predicted by temperature and the interaction between temperature and zooplankton density. We include uplooking acoustics data to complement our downlooking datasets, which provided evidence for potential bias in downlooking acoustic assessments of alewife due to high proportions of alewife found in the surface exclusion zone. Our approach combining several datasets provides a new perspective to understand summer diel distribution of alewife and the factors driving their distribution.