The goals of this study were to measure the relative levels of the α- and β-isoforms of myosin heavy chain (MHC-α and MHC-β, respectively) in multiple, specific regions of the adult rat heart and to determine whether age-dependent changes in isoform levels in different regions are uniform. Relative amounts of MHC-α and MHC-β were determined in right and left atria and left ventricular (LV) Purkinje fibers (PF), papillary muscles, trabeculae, and endo-, mid-, and epicardial regions at 2, 5, 10, 16, and 21 mo. PFs contained substantial amounts of myosin and were striated and capable of generating force and shortening on activation. Levels of MHC-β increased in all LV compartments with age, especially between 2 and 5 mo. There was more MHC-β in PFs than other LV sites. There were regional differences in the level of MHC-β throughout the LV at all ages, and the rates of change within regions differed. Ca2+-activated tension in PFs and trabeculae was compared at 2 and 22 mo. PF tension was less than trabecula tension, and this difference may be explained by differences in MHC content. Vmax and tension-generating ability in PFs decreased with age. Maximal tension generated by trabeculae did not change during aging. A large proportion of the increase in the level of MHC-β that is normally associated with aging occurs at a relatively early age in rat LV. PFs, with their small diameters and short diffusion distance, should be considered for skinned multicellular myocardial studies.