In RPE cells Ca²⁺ is involved in ROS phagocytosis, growth factor secretion, epithelial Cl^--transport and apoptosis. Thus a disturbance of the Ca²⁺-homeostasis in RPE cells may lead to serious functional impairments leading to cell death, and therefore participating in the genesis of age-related Macular Degeneration (AMD). This study showed that extracellular application of a sublethal dose of OH^-radicals (Fenton reaction) resulted in a biphasic [Ca²⁺]_i increase when using the Fura 2/AM method and calculating the ratio between the emission intensities at 340 and 380nm. The initial, transient peak increased to about 7% of the baseline value. This peak was independent of [Ca²⁺]_e and could be inhibited by adding 2mM Thapsigargin, suggesting a release of Ca²⁺ from intracellular Ca²⁺-stores. A second, continuous [Ca²⁺]_i increase was seen some time after terminating the OH^-application only when the first peak was present and the extracellular bath solution contained Ca²⁺. By blocking the L-type Ca²⁺-channel with 10mM Nifedipine and the reverse modus of the Sodium/Calcium Exchanger NCX1 with 10mM KB-R7943 we showed that NCX1 was partially involved in the second phase of the [Ca²⁺]_i rise. We conclude that extracellular OH^-leads to a Ca²⁺-activation from intracellular stores and this [Ca²⁺]_i elevation subsequently results in an influx of extracellular Ca²⁺.