Electrophysiological alternans is a beat-to-beat alternation of the action potential duration and/or Ca(2+) transient amplitude and is linked to ventricular arrhythmias. We investigated the significance of various rate parameters under different experimental conditions with respect to alternans incidence and the propensity for spiral wave formation. Voltage and Ca(2+) were optically mapped in monolayers of neonatal rat ventricular myocytes. Alternans did not occur at physiological temperature, but its incidence increased significantly at lowered temperatures. Pacing cycle length for spatially concordant alternans onset (PCL(C)), PCL for spatially discordant alternans onset (PCL(D)), and minimum cycle length for loss of 1:1 or 2:2 capture (MCL) also significantly increased with lower temperature but in a way such that the differences between PCL(C) and MCL and between PCL(D) and MCL widened. These results provided the rationale to identify the former difference as the alternans vulnerable window (AVW; in ms) and the latter difference as the discordant alternans vulnerable window (AVW(D); in ms). Computational simulations showed that interventions that widen AVW, including altered Ca(2+) cycling and enhanced K(+) currents, also promote alternans, regardless of whether PCL(C) or MCL increased or decreased. The simulation results were confirmed experimentally by addition of the ATP-sensitive K(+) channel agonist pinacidil. Mathematical analysis provided a theoretical basis linking the size of AVW to the incidence of alternans. Finally, experiments showed that the size of AVW(D) is related to the incidence of spatially discordant alternans and, additionally, to the incidence of spiral wave formation. In conclusion, vulnerable windows can be defined that are strongly correlated with alternans incidence, spatial discordance, and spiral wave formation.