Nicotine inhibits the activity of acetylcholinesterase (AChE) in vitro from brain and several cerebral structures. Thus, the aim of this study was to determine the ex vivo effect of nicotine on the activity from total brain, hippocampus, cerebral cortex, water and detergent soluble fractions of rats exposed to nicotine. The animals were kept in cages at room temperature. They had free access to water and food and were maintained on an inverted 10 h light 14 h dark cycle. They were treated with saline 0.9 % or nicotine 5mg/kg/day (s.c.) (5 consecutive doses at 8, 10, 12, 14 and 16:00 h) during 28 or 56 days. After this period, they were killed by decapitation and their brains were collected and hippocampus and cerebral cortex dissected. The hippocampus and cortex was homogenized in Tris-HCl buffer 10 mM, pH 7.2.  Aliquots were collected and the remaining homogenate was centrifuged at 14,500 g for 30 minutes at 4^oC. The supernatant was collected and considered as the water-soluble AChE (mainly G1 form). The pellet of the homogenate was suspended in a mixture of Triton X-100 and sodium phosphate buffer 50 mM pH 7.2. The suspension was stirred for 10 min at 10^oC. After this, it was centrifuged at 43,200 g for 30 min at 4^oC. The supernatant was taken as membrane-bound or Triton soluble enzyme (mainly G4 form). The residual pellet was suspended in sodium phosphate buffer 50 mM pH 7.2, and it was considered residual enzyme. The assay of acetylcholinesterase was performed by Ellman’s method. The results show that the AChE activity, for both periods of treatment, from total brain, cerebral cortex and hippocampus was not inhibited by nicotine. The detergent soluble fraction from hippocampus and cerebral cortex of rats treated with saline or nicotine, presented an AChE activity around 9 and 8 times higher than the water-soluble fraction, respectively. Besides, theses activities were not sensitive to nicotine in vivo. Hence, the absence of nicotine effect is not dependent on the molecular forms of the AChE.