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The factors that influence plankton distribution in lakes are currently widely debated. The primary objective of this study was to determine a combination of factors that influence the three-dimensional distribution patterns of both rotifer and crustacean communities in a pelagic ecosystem. We compared the abiotic (temperature, oxygen and nutrients) and biotic (phytoplankton) factors that affect the horizontal and vertical distribution of zooplankton in different habitat conditions in Lake Wigry. The results of our study indicate that the vertical gradient in a water profile is much more important for microcrustaceans than horizontal changes in environmental conditions, whereas Rotifera showed a strong spatial autocorrelation when connected with differences in trophic status. Generally, large zooplankton prefer cold, darker waters of the metalimnion-hypolimnion, while smaller zooplankton prefer the warm epilimnion. This niche segregation in water profiles promotes a large diversity of pelagic zooplankton. The vertical distribution of dominant Daphnia cucullata was strongly related to the phytoplankton distribution. Moreover, we found that the large-bodied Daphnia cucullata prefers the lower water layers, despite the presence of less optimal food resources, while smaller individuals clearly prefer the warm surface water with high quality resources.