We have investigated the effects of various mydriatic agents on the locomotion of polymorphonuclear leukocytes in vitro. Human as well as rat neutrophils showed a dose dependent increase of migration into micropore filters when tested against cyclopentolate hydrochloride at a dose range between 16 and 63 μg/ml. At higher doses (250 μg/ml), a complete inhibition of neutrophil migration was observed. A commercially available cyclopentolate hydrochloride preparation showed identical effects. Little or no changes in neutrophil locomotion were seen with atropine, homatropine, scopolamine or tropicamide when tested at the same concentration range. Since addition of cyclopentolate to either the lower or upper compartment of the multiwell chemotaxis chamber gave virtually the same results, it is assumed that the drug most likely induces a chemokinetic neutrophil response. However, an additional chemotactic effect cannot be excluded. These in vitro observations may help to explain an accidental observation in a patient with severe anterior uveitis who showed a massive, localized leukocyte accumulation on the corneal endothelium after contact with a cyclopentolate-soaked cotton pledget.