In the mid-1990s, biologists conducting assessments of fish stocks in Lake Ontario reported finding alewives Alosa pseudoharengus, rainbow smelt Osmerus mordax, and juvenile lake trout Salvelinus namaycush at greater depths than in the mid-1980s. To determine if depth distributions shifted coincident with the early 1990s colonization of Lake Ontario by exotic Dreissena mussels, we calculated mean depth of capture for each of the three species during trawl surveys conducted annually during 1978–1997 and examined the means for significant deviations from established patterns. We found that mean capture depth of alewives, rainbow smelt, and age-2 lake trout shifted deeper during the build up of the dreissenid population in Lake Ontario but that timing of the shift varied among seasons and species. Depth shifts occurred first for rainbow smelt and age-2 lake trout in June 1991. In 1992, alewives shifted deeper in June followed by age-2 lake trout in July–August. Finally, in 1993 and 1994, the distribution of lake trout and alewives shifted in April–May. Reasons why the three fishes moved to deeper water are not clear, but changes in distribution were not linked to temperature. Mean temperature of capture after the depth shift was significantly lower than before the depth shift except for alewives in April–May. Movement of alewives, rainbow smelt, and age-2 lake trout to colder, deeper water has the potential to alter growth and reproduction schedules by exposing the fish to different temperature regimes and to alter the food chain, increasing predation on Mysis relicta in deep water and decreasing alewife predation on lake trout fry over nearshore spawning grounds in spring.