Latanoprost, the active principle of Xalatan eye drops, is a prostaglandin F2alpha analogue in widespread use for the treatment of glaucoma. During chronic treatment with the drug, an increased pigmentation of the iris was observed in both primates and man. To gain an insight into the nature of this effect, we analyzed the stroma of the irides of cynomolgus monkeys subjected to 25-38 weeks of treatment. A highly sensitive procedure, based on chemical degradation by alkaline hydrogen peroxide oxidation, or hydriodic acid hydrolysis, was developed, which allowed eumelanin and pheomelanin analysis of a single iris at a time. Untreated monkey irides were found to be essentially pheomelanic, providing further support to the recently reported occurrence of these pigments in human irides. In the Latanoprost-treated eyes, the amount of eumelanin increased from three to sevenfold, while the variation of pheomelanin did not exceed 25%. The increase in eumelanin/pheomelanin ratio in the treated eyes, as compared with the contralateral control eyes, varied from three to fivefold, and the change was statistically significant (P < 0.01; t-test). Based on the results of parallel studies, showing that Latanoprost does not induce proliferation of iridial melanocytes, and that the other pigmented layers of the iris which do not contain melanocytes are not affected by the drug, it can be concluded that the observed effect is a result of a direct interaction with the melanogenic mechanism. This probably involves activation of tyrosinase, as suggested, to account for the stimulation of melanin synthesis by related compounds, including natural prostaglandins.