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Sprague-Dawley rats are often the background stock for transgenic rats, and rats from various sources may differ in their physical development and behavior. In 1990, the Institute of Cytology and Genetics in Novosibirsk obtained Sprague-Dawley rats from a commercial vendor and bred them in a closed colony. To study various aspects of the physical development and behavior of male F1 progeny of the Novosibirsk colony (Nov:SD) and commercial colony (Crl:SD) raised in identical environments, we evaluated body weight; food and water consumption; behavior in the elevated-plus maze (X maze), open field, free exploration paradigm, hole board, and the rotarod; and serotonin content in brain regions. Crl:SD rats were heavier and consumed more food than did the Nov:SD rats, which displayed a higher level of motor activity in all tests without displaying differences in anxiety-related behavior in the X maze or open-field test. In the free exploration paradigm, more Nov:SD rats explored the outside and started exploration earlier; they also were more active and showed less habituation in the hole-board test. Brain serotonin content was higher in the Crl:SD rats. In conclusion, prolonged isolated breeding of 2 stocks of Sprague-Dawley rats led to populations that differed in their exploratory and anxiety-related behavior, physical development, and serotonergic neurotransmission. Therefore, rats of the same stock but obtained from different breeders should be used with caution in research involving these measures.