The disease mechanisms associated with onset and secondary effects of rotavirus (RV) diarrhoea remain to be determined and may not be identical. In this study we investigated whether onset of RV diarrhoea is associated with increased intestinal permeability and/or motility. To study the transit time, fluorescent FITC-dextran was given to RV-infected adult and infant mice. Intestinal motility was also studied with an opioid receptor agonist (loperamide) and a muscarinic receptor antagonist (atropine). To investigate whether RV increases permeability at onset of diarrhoea, fluorescent 4- and 10-kDa dextran were given to infected and non-infected mice and fluorescence intensity measured subsequently in serum. RV increased transit time in adult and infant mice. Increased motility was detected as early as 6 hours post infection (h p.i) in adults and 24 h p.i. in pups, and persisted up to 72 h p.i in both models. Both loperamide and atropine decreased intestinal motility and attenuated diarrhoea. Analysis of passage of fluorescence dextran from the intestine into serum indicated unaffected intestinal permeability at onset of diarrhoea (24-48 h p.i.). We show that RV-induced diarrhoea is associated with increased intestinal motility via an activation of the myenteric nerve plexus, which in turn stimulates muscarinic receptors on intestinal smooth muscles.