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Introduction: The free exploratory paradigm is regarded as a reliable test for trait anxiety in mice but it may also be useful in rats. Previously, we showed that rat strains differ in their free exploration of novel areas, i.e. the surroundings of their familiar home cage when the lid was removed. Aim: Therefore, the purpose of the present study was to further examine strain, sex, and age differences in animals from different breeders in combination with pharmacological treatment designed to modify anxiety. Methods: In the present study free exploratory behaviour test was evaluated in Sprague Dawley and Wistar rats from different breeders. We assessed seasonal variation, habituation to the test, and the impact of gender and age on exploration. Furthermore, we monitored exploration following intraperitoneal diazepam, 8-OH-DPAT and caffeine administration. Parameters measured were latency to start exploring the outside of the cage, the percentage of rats that explored the outside, as well as the number of visits. Results: There was no seasonal variability in free exploratory behaviour. However, strains and sexes differed in the test results, though age-related differences had less impact. Diazepam (2mg/kg) and 8-OH-DPAT (30, 100 and 300μg/kg) decreased neophobia while caffeine (50mg/kg) increased the latency to explore the outside the next day. Discussion: The free exploratory behaviour test can be used as a simple and complementary test to study trait anxiety-related behaviour in rats.