Peptides derived from the precursor of A- and B-type natriuretic peptides (ANP and BNP) are powerful clinical markers of cardiac hypertrophy and dysfunction. It is known that many stimuli affecting the intracellular calcium concentration also induce ANP and BNP secretion. It was our intention to study the mechanisms by which calcium regulates the secretion of ANP and BNP. The effects of pacing and calcium-calmodulin kinase II (CaMKII) activity on NP secretion were studied in isolated perfused rat atria and in cultured rat neonatal cardiomyocytes. In isolated rat atrium pacing induced an increase in diastolic, systolic and averaged [Ca²⁺]i and a frequency-dependent increase in the secretion of both ANP and BNP. The molar ratio of the secreted NPs (ANP/BNP) remained nearly constant ( 1000) at all the pacing frequencies tested (1 Hz, 3 Hz, 6 Hz and 8 Hz). Calmodulin kinase II inhibitor KN-93 (3 µM) did not affect [Ca²⁺]i, but showed a frequency-dependent inhibitory effect on ANP and BNP secretion without a change in ANP/BNP-ratio. In the neonatal cardiomyocytes, KN-93 (3 µM) suppressed the secretion and gene expression of both ANP and BNP. Overexpression of constitutively active (T286D) or nuclear (_B) CaMKII induced an increase in ANP and BNP gene expression. The results indicate that the calcium dependent secretion and gene expression of A- and B-type natriuretic peptides are similarly regulated by calmodulin kinase II-dependent mechanisms. This is a plausible mechanism contributing to exercise-induced natriuretic peptide secretion and the augmented secretion in heart dysfunction due to impaired calcium handling.