Hyperosmolality has been known to increase ANP release. However, its physiological role in the regulation of atrial myocytic ANP release and the mechanism by which hyperosmolality increases ANP release are to be defined. The purpose of the present study was to define these questions. Experiments were performed in perfused beating rabbit atria. Hyperosmolality increased atrial ANP release, cAMP efflux and atrial dynamics in a concentration-dependent manner. The osmolality threshold for the increase in ANP release was as low as 10 mOsm/kg above the basal levels by about 3% (1.55 ±1.71, 17.19 ± 3.11, 23.15 ± 5.49, 54.04 ± 11.98 and 62.00 ± 13.48% for mannitol 10, 20, 30, 60 and 100 mM, respectively, all P < 0.01). Blockade of sarcolemmal L-type Ca²⁺ channel activity which increased ANP release attenuated hyperosmolality-induced increases in ANP release (-13.58 ± 4.68% vs. 62.00 ± 13.48%, P < 0.001) and cAMP efflux but not atrial dynamics. Blockade of the Ca²⁺ release from the sarcoplasmic reticulum which increased ANP release attenuated hyperosmolality-induced increase in ANP release (13.44 ± 7.47% vs. 62.00 ± 13.48%, P < 0.01) and dynamics but not cAMP efflux. Blockades of Na⁺-K⁺-2Cl^- cotransporter, Na⁺-H⁺ exchanger and Na⁺-Ca²⁺ exchanger had no effect on hyperosmolality-induced increase in ANP release. The present study suggests that hyperosmolaity regulates atrial myocytic ANP release, and that the mechanism by which hyperosmolality activates ANP release is closely related to the cross-talk between the sarcolemmal L-type Ca²⁺ channel activity and sarcoplasmic reticulum Ca²⁺ release, possibly inactivation of the L-type Ca²⁺ channels.