Modafinil is a wakefulness-promoting agent with possible beneficial effects for the management of addiction and in psychiatric conditions, but also with abuse potential of its own. The mechanism of action of modafinil remains unclear. We studied pharmacological mechanisms in the effect of modafinil on prepulse inhibition (PPI), a model of sensorimotor gating. Mice were tested in automated startle boxes after administration of modafinil and antagonist drugs. Oral administration of 100mg/kg of modafinil, but not lower doses, caused a significant reduction of PPI in C57Bl/6 mice, but not Balb/c mice. This effect of modafinil could be blocked by co-treatment with the dopamine D(2) receptor antagonist, haloperidol, and the serotonin (5-HT) 2A receptor antagonist, ketanserin, but not the 5-HT(1A) receptor antagonist, WAY100,635. At 30mg/kg, which did not influence PPI, modafinil inhibited PPI disruption caused by the dopamine transporter inhibitor, GBR12909. There was no interaction between modafinil and the serotonin transporter inhibitor, fluoxetine. There were no consistent effects of modafinil on startle amplitude. These results show that oral modafinil treatment may cause disruption of PPI in mice. This effect was strain-dependent, involving dopamine D(2) and 5-HT(2A) receptor activation, and was likely mediated by an interaction with the dopamine transporter. These results extend our insight into the behavioral effects of modafinil and could be of importance for the clinical use of this agent as they may indicate an increased risk of side-effects in conditions where PPI is already reduced, such as in schizophrenia and bipolar disorder.