The spatial and dynamic properties of ventricular fibrillation (VF) may be random or related to cellular electrical properties of the normal heart. Local activation intervals (AIs) in VF may depend on the local refractory period (RP), and sustained VF may require a steep action potential (AP) restitution curve. In guinea pig hearts, AP durations (APDs) and RPs on the epicardium are shorter at the apex and progressively longer toward the base, producing gradients of RPs that may influence the spatial organization of VF. In the present study, the influence of APDs on VF dynamics is investigated in perfused guinea pig hearts stained with a voltage-sensitive dye by comparing APD gradients to the dynamics of VF elicited by burst pacing. In VF, AIs had no clear periodicity, but average AIs were shorter at the apex (57.5+/-8.1 ms) than the base (76.1+/-1.5 ms, n=6, P<0.05) and had gradients similar to APD gradients (correlation coefficient 0.71+/-0.04). Analysis of local velocity vectors showed no preferential directions, and fast Fourier transform (FFT) power spectra were broad (10 to 24 Hz) with multiple peaks (n=6). However, the selective inhibition of delayed K(+) rectifying currents, I(Kr) (E4031; 0.5 micromol/L, n=3), shifted FFT spectra from complex to a lower dominant frequency (10 Hz) and altered repolarization but retained the correlation between mean AIs and RPs. Thus, VF dynamics are consistent with a multiple wave-make and wave-break mechanism, and the local RP influences VF dynamics by limiting the range of VF frequencies and AIs at each site. The full text of this article is available at http://www.circresaha.org.