Medetomidine is a new antifouling agent, and its effects in non-target aquatic organisms have been investigated. Earlier short-term studies in fish have shown a skin lightening response to medetomidine, but effects after chronic exposure have not been studied. In fish, the dark pigment melanin is contained within specialized cells, melanophores. Medetomidine binds to the melanophore alpha2-adrenoceptor, which stimulates pigment aggregation resulting in the light appearance. In the present study, rainbow trout (Oncorhynchus mykiss) was long-term exposed to 0.5 and 5.0 nM of medetomidine via water for 54 d. The fish were then photographed for paleness quantification and the images were analyzed using ImageJ analysis software. Additionally, scales were removed and used for in vitro function studies of the melanophores, monitoring the response to melanophore stimulating hormone (MSH) and subsequent medetomidine addition. The number of melanophores was also investigated. As a result of the medetomidine exposure, fish from the 5 nM treatment were significantly paler than control fish and the melanophores from these fishes were also more aggregated. Melanophores from all the treatments were functional, responding to MSH by dispersion and to subsequent medetomidine by aggregation. However, the results indicate a difference in sensitivity among treatments. The number of melanophores in the scales did not change significantly after long term exposure to medetomidine. These results suggest that the observed paleness may be reversible, even after chronic exposure.