Myocardial slices, also known as "cardiac tissue slices" or "organotypic heart slices," are ultrathin (100-400 μm) slices of living adult ventricular myocardium prepared using a high-precision vibratome. They are a model of intermediate complexity as they retain the native multicellularity, architecture, and physiology of the heart, while their thinness ensures adequate oxygen and metabolic substrate diffusion in vitro. Myocardial slices can be produced from a variety of animal models and human biopsies, thus providing a representative human in vitro platform for translational cardiovascular research. In this review, we compare myocardial slices to other in vitro models and highlight some of the unique advantages provided by this platform. Additionally, we discuss the work performed in our laboratory to optimize myocardial slice preparation methodology, which resulted in highly viable myocardial slices from both large and small mammalian hearts with only 2-3% cardiomyocyte damage and preserved structure and function. Applications of myocardial slices span both basic and translational cardiovascular science. Our laboratory has utilized myocardial slices for the investigation of cardiac multicellularity, visualizing 3D collagen distribution and micro/macrovascular networks using tissue clearing protocols and investigating the effects of novel conductive biomaterials on cardiac physiology. Myocardial slices have been widely used for pharmacological testing. Finally, the current challenges and future directions for the technology are discussed.