Particles composed of inorganic, organic and/or biological materials occur in both natural water bodies and aquaculture facilities. They are expected to affect fish health through a direct chemical, mechanical and biological interaction with gills during ventilation but the nature of the reactions and the relative importance of mechanical versus chemical and biological stimulation are unknown. The present work presents an immune gene expression method for evaluation of gill disturbance and sets a baseline for the mechanical influence on fish gills of chemically inert spherical particles. The method may be applied to investigate particle impact at different combinations of temperature, fish size, water quality and particle composition. Spherical polystyrene particles (diameters 0.2 μm, 1 μm, 20 μm, 40 μm and 90 μm) were adopted as the particle model and the rainbow trout (Oncorhynchus mykiss) fingerlings and wildtype zebrafish (Danio rerio) as fish models. After 2 h particle exposure (concentration 2 × 10⁵ particles/L), particles present in different gill compartments were recorded and gill responses evaluated by quantitative realtime PCR focusing on innate and adaptive immune genes. Particles measuring 20 μm and 40 μm were more frequently located in rainbow trout gills compared to 90 μm beads. Fish gills colliding with inert spherical particles may not necessarily and generally initiate inflammatory reactions. The ifnγ gene showed upregulation in rainbow trout gills (exposed to 0.2 μm and 40 μm beads) and in zebrafish gills (exposed to 1 μm and 90 μm beads). The genes il1β and igm in zebrafish gills (exposed to 1 μm particles) and s100a (exposure to 0.2 μm beads) were also upregulated. In rainbow trout down-regulation was noted with regard to il1β (bead size 1 μm), s100a (bead size 40 μm) and saa (1, 40 and 90 μm). In zebrafish gills down-regulation was found for the il8gene (0.2 and 20 μm). The effect of particle exposure is discussed with regard to shape and chemical/biological composition of particles in water and to abiotic parameters including temperature.