Pulmonary surfactant is secreted via exocytosis of lamellar bodies (LBs) by alveolar type II cells. Here we analyzed the dependence of LB exocytosis on intracellular Ca²⁺concentration ([Ca²⁺]_i). In fura 2-loaded cells, [Ca²⁺]_iwas selectively elevated by flash photolysis of a cell-permeant caged Ca²⁺ compound (o-nitrophenyl EGTA-AM) or by gradually enhancing cellular Ca²⁺influx. Simultaneously, surfactant secretion by single cells was analyzed with the fluorescent dye FM 1-43, enabling detection of exocytotic events with a high temporal resolution (T. Haller, J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. Proc. Natl. Acad. Sci. USA 95: 1579–1584, 1998). Exocytosis was initiated at a threshold concentration near 320 nmol/l with both instantaneous or gradual [Ca²⁺]_ielevations. The exocytotic response to flash photolysis was highest during the first minute after the rise in [Ca²⁺]_iand thus almost identical to purinoceptor stimulation by ATP. Correspondingly, the effects of ATP on initial secretion could be sufficiently explained by its ability to mobilize Ca²⁺. This was further demonstrated by the fact that exocytosis is significantly blocked by suppression of the ATP-induced Ca²⁺ signal below ∼300 nmol/l. Our results suggest a highly Ca²⁺-sensitive step in LB exocytosis.