To characterize the effects of the cellular events associated with contraction on atrial natriuretic factor (ANF) secretion, primary neonatal rat atrial myocytes were electrically paced to contract while being monitored for ANF release, cytoplasmic calcium, phosphoinositide hydrolysis, and protein kinase C activation. Similar measurements were also carried out in the presence of endothelin-1 (ET) for comparison of contraction-related and hormone-stimulated ANF secretion. Pacing (6-8 Hz) immediately increased ANF secretion by 3-5-fold and the time-averaged cytoplasmic calcium concentration (as monitored with indo-1 fluorescence) varied with pace frequency in a similar manner, suggesting that cytoplasmic calcium may play a key role in pace-induced ANF secretion. Furthermore, nifedipine and ryanodine, which inhibited the contractile calcium transients, inhibited pace-induced ANF release, whereas Bay K 8644 increased both the calcium transients and ANF secretion. Pace-induced ANF release was also completely inhibited by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK) but was not inhibited by chelerythrine, a protein kinase C-selective inhibitor. Pace-induced ANF release averaged 40% of that elicited by ET which is known to require both PKC and CaMK for maximal effects on ANF secretion. The effects of pacing and ET on ANF secretion were approximately additive. In contrast to pacing, ET strongly stimulated phosphoinositide hydrolysis, activated PKC, and did not increase cytoplasmic calcium. Thus, regulation of ANF secretion by contraction rate depends primarily on the contractile calcium transients and CaMK and is independent of PKC.