The time course and magnitude of the Ca2+ fluxes underlying spontaneous Ca2+ waves in single permeabilized ventricular cardiomyocytes were derived from confocal Fluo-5F fluorescence signals. Peak. ux rates via the sarcoplasmic reticulum (SR) release channel (RyR2) and the SR Ca2+ ATPase (SERCA) were not constant across a range of cellular [Ca2+] values. The Ca2+ affinity (K-mf) and maximum turnover rate (V-max) of SERCA and the peak permeability of the RyR2-mediated Ca2+ release pathway increased at higher cellular [Ca2+] loads. This information was used to create a computational model of the Ca2+ wave, which predicted the time course and frequency dependence of Ca2+ waves over a range of cellular Ca 21 loads. Incubation of cardiomyocytes with the Ca2+ calmodulin (CaM) kinase inhibitor autocamtide-2-related inhibitory peptide (300 nM, 30 mins) significantly reduced the frequency of the Ca2+ waves at high Ca2+ loads. Analysis of the Ca2+ fluxes suggests that inhibition of CaM kinase prevented the increases in SERCA Vmax and peak RyR2 release. ux observed at high cellular [ Ca 21]. These data support the view that modi. cation of activity of SERCA and RyR2 via a CaM kinase sensitive process occurs at higher cellular Ca2+ loads to increase the maximum frequency of spontaneous Ca2+ waves.