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It has been reported that the use of certain stimuli can lead to anxiety-like behavior in zebrafish. Moreover, visual stimulation of zebrafish is becoming a popular tool. Here we evaluated the effects of six colors combinations and alprazolam, a benzodiazepine which is widely used in the treatment of anxiety disorders, on the behavior of adult zebrafish in a two-chambered apparatus, which chambers were illuminated by red/yellow, green/blue, red/green, green/yellow, red/blue and blue/yellow light. The following parameters were measured: time spent in the zone, number of entries to the zone, time of freezing, distance traveled and average speed in the zone. We report that the adult zebrafish spent more time in the red zone compared to yellow or green as well as in the yellow or blue compared to green. The zebrafish displayed a concomitant increase in time freezing in the red zone compared to yellow or green as well as in the yellow or blue compared to green. Moreover, average speed was decreased in the red zone compared to yellow or green and in the yellow zone compared to green. Treatment with alprazolam significantly affected the behavior of the zebrafish, e.g., following alprazolam administration time spent in the zone and time freezing were longer in the green zone than in red. Based on these observations, we suggest that light color combinations could be effective to manipulate zebrafish behavior and could be useful in neuropsychopharmacological studies, perhaps to study anxiety-like behavior and the effects of anxiolytic agents.Combination of colors as stimuli enables to manipulate zebrafish behavior.Treatment with alprazolam significantly affected the behavior of the zebrafish.Color stimulation can be useful to test the effects of drugs on behavior.