Introduction: The action potential duration (APD), measured from optical action potentials (OAPs), is expected to approximate the activation-recovery interval (ARI) from unipolar electrograms (UEGs). However, this relationship can vary by species, myocardial surface (endo vs. epi), and disease state. In heart failure, increased ARI variability has been linked to a higher risk of arrhythmias. This study investigates these factors using an endo-epicardial approach in porcine and human hearts. Methods: Three pig hearts and two human hearts (VCU IRB approved) were prepared using an open-chamber Welch setup, perfused with Tyrode's solution, and stained with JPW-6003. Optical signals were recorded from both surfaces using two high-speed cameras with 660 nm LEDs. Simultaneously, custom-made 4×4 transparent electrode arrays (1 cm spacing) recorded UEGs from the same regions. Pacing was performed using decremental PCL protocols. Local activation time (LAT) was defined as the minimum dV/dt in UEGs and the 50% upstroke in OAPs. APD80 was calculated from LAT to 80% repolarization. ARI was measured from LAT to the maximal T-wave slope (Wyatt method), regardless of polarity. Analysis included three beats per position from all electrodes and adjacent pixels. Results: In this representative dataset, ARI values were consistently lower than APD80 across species and surfaces. Endocardial measurements showed smaller differences in pigs and higher variability in humans. ARI presented greater beat-to-beat fluctuation, indicating higher temporal consistency in OAP-derived APD. Conclusion: The observed discrepancies between ARI and APD80—both in absolute values and variability—suggest that ARI may underestimate true repolarization duration under specific conditions. Simultaneous optical-electrical mapping may improve the characterization of ventricular repolarization dynamics.