The purpose of the study was to evaluate the behavior of shelter and the activity of cerebral acetylcholinesterase (AChE-cerebral, EC: 3.1.1.7) in the species Gambusia punctata (Poey, 1854) by bioavailable lead. The water matrix of the San Juan ecosystem of Santiago de Cuba-Cuba was sampled by selecting a composite sample (1: 1: 1) from three points corresponding to the lower part of this ecosystem. Then the reaction time-refuge of G. punctata, the activity of the AChE-brain by action of the bioavailable lead concentration for the control and experimental treatment group was tested for 21 days at 25 °C. The reaction-refuge time was delayed and the enzymatic activity of the AChE-brain was elevated. A greater bioaccumulation of brain Pb was observed in the experimental group. It is concluded that the refuge behavior was possibly reduced due to the increase in the activity of the AChE-cerebral enzyme, according to bioaccumulation of the Pb, indicating a biological anomaly.