A combination of early neurodevelopmental disruptions and young-adult cannabis use may lead to the development of neuropsychiatric disorders. The aim of this study was to investigate in adult Wistar rats (12-14 weeks of age) the long-term 'two hit' behavioural effects of chronic young-adult treatment with the cannabinoid receptor agonist, CP55,940 (0.2 mg/kg, 8-10 weeks of age) in combination with maternal separation (MS) (3 h every day from postnatal days 2-14). Two weeks after chronic CP55,940 treatment had ceased, baseline locomotor activity was reduced in male, but not female rats and irrespective of MS. In male rats only, the combination of MS and cannabinoid exposure, but not either 'hit' alone, induced a significant decrease in sucrose preference. In contrast, in male rats both MS and CP55,940 treatment reduced time spent on the open arms of the plus maze or centre time in the open field and this was most pronounced after a combination of these 'hits'. Prepulse inhibition was reduced by MS in both sexes but there was no additional effect of CP55,940 treatment. Memory performance in the Y-maze and novel object recognition test was not affected by either of the two 'hits'. These results indicate that early developmental disruptions and young-adult cannabis use on their own or in combination can differentially and sex-specifically affect behaviours related to neuropsychiatric disorders.