Environmental stressors can substantially affect the adaptive response of rats to novelty in a sexually dimorphic manner. Gender-related differences are also observed in neurochemical and behavioural patterns of adult rats following prenatal exposure to diazepam (DZ). In the present study the behavioural reactivity to novelty is investigated in open field (OF) and in acoustic startle reflex (ASR) tests, in non handled (NH), short-lasting handled (SLH) and long-lasting handled (LLH) adult male and female rats prenatally exposed to DZ. A single daily s.c. injection of DZ (1.5 mg/kg) over gestation days 14-20 decreases GABA/BDZ receptor function in both sexes, as shown by the decreased electrographic hippocampal response to DZ and the increased response to picrotoxin, after intra-locus coeruleus injection of the two compounds. In OF NH DZ-exposed males display a lower total distance travelled (TDT), a higher rearing frequency (RF) and a greater number of transitions in the centre of the arena (CNT) compared to NH rats prenatally exposed to vehicle. Conversely, NH DZ-exposed females show slight changes in TDT and RF and a greater reduction in CNT and in the amount of time spent in the centre of the arena (CAT). These effects are associated with an increase in the peak amplitude of the ASR in both sexes. Short-lasting handling slightly influences DZ-evoked effects in animals of both sexes. In DZ-exposed males long-lasting handling attenuates the reduction in TDT and the enhancement in RF, prevents the increase in CNT and reduces the peak amplitude of ASR. In DZ-exposed females, long-lasting handling increases TDT and RF, induces a lower avoidance of the centre of the arena, and does not modify the peak amplitude of ASR, when compared to controls. These findings indicate that prenatal exposure to DZ differently affects behavioural reactivity in adult male and female rats, and suggest that a long-lasting handling is able to attenuate some behavioural deficits induced by prenatal DZ exposure.