Several characteristics warrant the zebrafish a refining animal model for toxicity testing in rodents, thereby contributing to the 3R principles (Replacement, Reduction, and Refinement) in animal testing, e.g. its small size, ease of obtaining a high number of progeny, external fertilization, transparency and rapid development of the embryo, and a basic understanding of its gene function and physiology. In this context we explored the motor activity pattern of zebrafish larvae, using a 96-well microtiter plate and a video-tracking system. Effects of induced light and darkness on locomotion of zebrafish larvae of different wild-type strains and ages (AB and TL, 5, 6 and 7 dpf; n = 25/group) were studied. Locomotion was also measured in zebrafish larvae after exposure to different concentrations of ethanol (0; 0.5; 1; 2 and 4%) (AB and TL strain, 6 dpf; n = 19/group). Zebrafish larvae showed a relatively high swimming activity in darkness when compared to the activity in light. Small differences were found between wild-type strains and/or age. Ethanol exposure resulted in hyperactivity (0.5-2%) and in hypo-activity (4%). In addition, the limitations and/or relevance of the parameters distance moved, duration of movements and velocity are exemplified and discussed. Together, the results support the suggestion that zebrafish may act as an animal refining alternative for toxicity testing in rodents provided internal and external environmental stimuli are controlled. As such, light, age and strain differences must be taken into account.