This study was undertaken in order to determine the relative extent to which amphetamine-induced mydriasis is of central nervous system (CNS) or of peripheral origin. Anesthetized cats were administered cumulative dosages of amphetamine given either intravenously (0.05-3.2 mg/kg) or directly into the third ventricle of the brain (500 micrograms i.c.v.). Pupillary dilation and ciliary nerve activity were monitored. In cats with only parasympathetic tone to the iris intact, i.v. amphetamine produced a dose-dependent mydriasis and decrease of ciliary nerve activity. Treatment with yohimbine (0.5 mg/kg i.v.), before amphetamine administration, blocked the amphetamine-induced reduction of parasympathetic nerve activity and partially antagonized the pupillary dilation. Both yohimbine pretreatment and CNS monoamine depletion (with reserpine 5 mg/kg i.p. and alpha-methyl-p-tyrosine 2 x 300 mg/kg i.p.) prevented the mydriatic and ciliary nerve activity lowering effects of i.c.v. amphetamine. These results suggest that amphetamine produces mydriasis in the cat primarily by means of CNS inhibition of tonic outflow from the oculomotor nucleus and to only a minor extent by acting as a peripheral sympathomimetic. This conclusion is consistent with the hypothesis that release of norepinephrine within the CNS inhibits tonic parasympathetic outflow to the iris.