BACKGROUND Pharmacologic testing by indirect acting sympathomimetics like hydroxyamphetamine may determine the site of the lesion in Horner's syndrome. Pholedrine is chemically similar to hydroxyamphetamine. Therefore we examined if it shows the same effects in normal subjects and in patients with Horner's syndrome. METHODS Pupil diameter was measured by means of standardized photography before and after single and with different intervals repeated administration of pholedrine eye drops in normal subjects. In 18 patients with Horner's syndrome and known hydroxyamphetamine test results, a pholedrine test was carried out analogous to the hydroxyamphetamine test. RESULTS Pholedrine dilates the normal pupil by 2.2 mm (mean). It acts at the longest 8-10 hours with maximal effect between 20 and 90 minutes. After this period its effect decreases rapidly. It acts independently from age and from baseline pupil diameter. Given repeatedly the second administration reaches the same mydriatic effect as the first only if the interval between both applications is 72 hours or more. This is because it needs some time to refill the noradrenaline stores in the sympathetic neuron innervating the dilator muscle of the pupil. In Horner's syndrome pholedrine dilates the involved pupil only minimally in case of a postganglionic lesion, and in case of a preganglionic lesion it dilates the pupil even slightly more than the normal fellow pupil. It shows the same effect as hydroxyamphetamine. There are only few side effects. CONCLUSIONS Pholedrine is a substitute for hydroxyamphetamine to localize the site of the lesion in patients with Horner's syndrome.