How microplastics transit within aquatic ecosystems and partition among environmental compartments is not fully understood. To increase understanding, we added microplastic fragments ranging in buoyancy (positive: polyethylene (PE), neutral: polystyrene (PS), negative: polyethylene terephthalate (PET)) and size (∼30 to 1400 μm) to surface waters of closed-bottom, in-lake mesocosms (10 m diameter, 2 m depth). To assess residence time, we measured microplastics in surface waters and the water column over a 9-week period. To measure fate, we measured microplastics in the surface water, water column, bottom detritus, and biota (biofilm on the walls, zooplankton, fish) at 9 weeks. The residence times of microplastics were longer at the surface than in the water column, with less dense and smaller particles having the longest residence times. After 9 weeks, nearly all microplastics were on the bottom, with only 3% on the surface, 0.4% in the water column, 2% in biofilm, and <
0.01% in zooplankton and fish. The surface water and biofilm on the walls were larger reservoirs than the water column, suggesting that surface microlayers and biofilm on hard substrates are important, yet overlooked, reservoirs of microplastics in aquatic ecosystems. Results inform future hypotheses relevant to monitoring programs and risk assessments.