The 96‐h median lethal concentration for profenofos in the Australian eastern rainbow fish (Melanotaenia duboulayi) was 0.9 mg/L, corresponding with an 83% reduction in acetylcholinesterase (AChE) activity in fish head. This is an order of magnitude less sensitive than the figure reported for crucian carp (Carassius carassius). Higher lethal body burdens were found in rainbow fish exposed to high concentrations of profenofos but the inhibition of AChE activity in these fish was less than in those killed by longer, normally sublethal, exposures. During sublethal exposure to 10 μg/L for 10 d, the fish accumulated profenofos residues of 5.3 mg/kg associated with a 70% reduction in AChE activity in fish head. Decreases in food intake, food conversion efficiency, and growth were observed at 10 d and significant loss of weight was noted at 21 d. These were associated with markedly increased swimming activity and response to light. A clear relationship existed between the depression of AChE activity and observed behavioral responses, such as swimming activity and food intake.