Previous studies have postulated an important role for the inwardly rectifying potassium current (I(K1)) in controlling the dynamics of electrophysiological spiral waves responsible for ventricular tachycardia and fibrillation. In this study, we developed a novel tissue model of cultured neonatal rat ventricular myocytes (NRVMs) with uniform or heterogeneous Kir2.1expression achieved by lentiviral transfer to elucidate the role of I(K1) in cardiac arrhythmogenesis. Kir2.1-overexpressed NRVMs showed increased I(K1) density, hyperpolarized resting membrane potential, and increased action potential upstroke velocity compared with green fluorescent protein-transduced NRVMs. Opposite results were observed in Kir2.1-suppressed NRVMs. Optical mapping of uniformly Kir2.1 gene-modified monolayers showed altered conduction velocity and action potential duration compared with nontransduced and empty vector-transduced monolayers, but functional reentrant waves could not be induced. In monolayers with an island of altered Kir2.1 expression, conduction velocity and action potential duration of the locally transduced and nontransduced regions were similar to those of the uniformly transduced and nontransduced monolayers, respectively, and functional reentrant waves could be induced. The waves were anchored to islands of Kir2.1 overexpression and remained stable but dropped in frequency and meandered away from islands of Kir2.1 suppression. In monolayers with an inverse pattern of I(K1) heterogeneity, stable high frequency spiral waves were present with I(K1) overexpression, whereas lower frequency, meandering spiral waves were observed with I(K1) suppression. Our study provides direct evidence for the contribution of I(K1) heterogeneity and level to the genesis and stability of spiral waves and highlights the potential importance of I(K1) as an antiarrhythmia target.