Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac repair, but more information is required as to their electrical behavior in vivo. hESC-CMs expressing the protein calcium sensor GCaMP3 provide a graft autonomous reporter of activation, which we have used to show hESC-CM grafts can couple with host myocardium in injured hearts. When we sought to assess host-graft electrical interactions by optical voltage mapping, we found the commonly used lipophilic voltage dyes RH237 and di-4-ANEPPS label host but not graft tissue. We hypothesized the water-soluble voltage dye di-2-ANEPEQ could overcome this limitation and efficiently label both host and graft. After confirming good spectral separation of GCaMP3 and di-2-ANEPEQ signals by spectrofluorimetry and confocal spectral imaging, we transplanted 1x10^8 GCaMP3+ hESC-CMs into guinea pig hearts (n=6) at 4 weeks following cardiac injury and then imaged engrafted hearts at 2 weeks post-transplantation with a dual-channel CCD-based system. We found a differential time-course of di-2-ANEPEQ labeling between host and graft tissues, with stable optical action potentials (APs) obtained in host and graft tissue after ~4 and ~13 minutes of dye perfusion, respectively. This differential labeling kinetics, which was also observed on washout, presumably reflects sluggish graft perfusion and provides another tool for distinguishing host and graft signals. No regions of 1:1 host-graft coupling were identified, and graft tissue had spontaneous rates from 0.1-2 Hz and long optical AP durations from 500-1090 ms. Activation maps based on di-2-ANEPEQ and GCaMP3 signals indicated relatively slow conduction velocities in graft tissue, as well as patterns of propagation that commonly occurred along a vector distinct from that in host tissue. These imaging studies reveal multiple potentially pro-arrhythmic properties in hESC-CM graft tissue including slow propagation, ultralong AP duration, as well as aberrant patterns of activation that can vary from beat to beat. We conclude the electrical behavior of both graft and host myocardium can be reliably assessed by the simultaneous imaging of a graft-autonomous fluorescent reporter of activation and a water-soluble voltage dye.