Papillary muscles (PMs) are known triggers of ventricular arrhythmia, especially nonsustained ventricular tachycardia (VT) and sustained recurrent VT, and may play a role as triggers of ventricular fibrillation. Catheter ablation of PM is challenging and results in high VT recurrence rates. Clinical characterization of PM electrophysiology is challenging due to the limited spatial resolution of electrode arrays, motion, and distal fields. Thus, ablation procedures are limited in two ways: inaccurate determination of target ablation zones and inaccurate characterization of lesion profiles. Characterization of PM before and after ablation can be accomplished with deep infrared optical mapping toward a better understanding of the role of the PM. Using optical mapping and near-infrared voltage-sensitive fluorescent dyes, transmembrane potentials were recorded from the endocardial side of a live explanted human right ventricle free-wall (transplant recipient). We paced the myocardium at different cycle lengths (CLs) before and after ablation, and transmembrane potential maps were obtained using excitation light bands of different penetration depths to characterize the ablated zones transmurally. The PM exhibited alternans in action potential duration (APD) at longer CLs than wall tissue in non-ablated tissue, indicating a highly heterogeneous myocardium. The figure shows relative conduction velocity maps for subsequent beats before and after ablation. While dissection of the tissue appeared to show a transmural burn, optical mapping with different penetration depths, from green (surface) and near-infrared (NI, transmural) illumination, revealed that the transmural electrophysiology profile was ablated, only at the surface. The presence of conduction velocity alternans using NI light indicated incomplete ablation at the deeper subsurfaces, even though it appeared burnt during dissection. Contemporary electro-mapping systems used in clinics often achieve limited electrophysiological characterization in the depth of ablated areas. Such measurements would yield results similar to optical mapping with the green light excitation band (surface only), suggesting successful ablation that may be incomplete and may result in recurrent VT. We demonstrate that optical mapping with NI light is a useful tool for quantitative characterization of ablation parameters.