Iron is one the most abundant metals on the earth being essential for living organisms even though its free form can be toxic. The overload of this metal may be related with some disorders, like Alzheimer and Parkinson diseases, and hemochromatosis in the liver. The aim of the present study was to evaluate the effects of iron on acetylcholinesterase (AChE) activity in brain and liver of zebrafish and to investigate the possible correlation with the iron content in these tissues. Different corresponding concentrations of iron were tested using in vitro (0.018, 0.268, and 2.6 mM) and in vivo (1, 15, and 150 mg/l) assays. The in vitro studies showed that iron promoted a significant increase in AChE activity in brain (52%) and liver (53%) at the higher concentration (2.6 mM). In the in vivo assays, a significant increase in this enzyme activity was observed in the presence of 15 mg/l in both, brain (62%) and liver tissue (70%). Semiquantitative RT-PCR did not reveal significant changes in acetylthiocholinesterase mRNA levels. Moreover, we observed that iron content was significantly increased in liver tissue when exposed to 15 (226%) and 150 mg/l (200%). These results indicate that iron can promote significant alterations in AChE activity which probably is not directly related to the iron content in zebrafish tissues.