This study was conducted in order to determine how various technical and biological factors affect brain cholinesterase (ChE) activity of rainbow trout Salmo gairdneri. Brain ChE activity increased in proportion to increasing temperature to 20°C, but at higher temperatures, the ChE activity increased at a slower rate and was absent at 45°C. Brain ChE activity remained stable for a long time when the rainbow trout were stored at −20°C or lower, and short (2-d) storage at 4°C was not detrimental; dead fish left 1 d at ambient water temperature also did not have lower ChE activities. Habitat temperature did not affect brain ChE activity significantly. Small rainbow trout had markedly higher brain ChE activities than large fish.