The effects of a daily injection of the opioid antagonist naltrexone (NALTX, 1 mg/kg SC), from birth to weaning day, on several behavioral parameters were investigated in adult rats of both genders. This work uses three behavioral tests. The use of the open-field test in addition to the holeboard and plus-maze tests allows the measurement of complementary parameters, and provided further information on diverse components of behavior. The effect of repeated testing (habituation) over 3 consecutive days of open-field testing was also assessed. In accordance with previous reports, female rats showed higher locomotor activity and exploration and a lower level of emotionality than their male counterparts. The response of the animals to repeated testing seemed to be both sex and treatment dependent. In both males and females, NALTX treatment during the preweanling period did not affect locomotor activity, but increased the animals' levels of anxiety and emotionality and decreased exploratory behavior. Our results indicate that neonatal opioids may play an important role in the development of behavioral responses in the adult.