This chapter describes methods to investigate mammalian cardiac channel properties at the single-channel level. Cell isolation is performed from adult heart by enzymatic digestion using the Langendorff apparatus. Isolation proceeding is suitable for rabbit, rat, and mouse hearts. Also, isolation of human atrial cardiomyocytes is described. In freshly isolated cells or cells maintained in primary culture, the single-channel variants of the patch-clamp technique (cell-attached, inside-out, and outside-out) are used to investigate channel properties. Biophysical properties such as conductance and ionic selectivity are determined. Also, regulations by extracellular and intracellular mechanisms are investigated. To illustrate the study, the author provides an example by the characterization of a calcium-activated nonselective cation channel (TRPM4).