Intact isolated cardiomyocytes are used to investigate the molecular mechanisms of contraction and to screen new therapeutic compounds. Typically, these studies require either daily isolation or culture of cardiomyocytes, which either costs investigators time and animals or alters myocyte function and morphology. Recently, investigators reported preserving canine cardiomyocyte function using N-benzyl-p-toluene sulphonamide (BTS), a skeletal muscle myosin ATPase inhibitor. We investigated the preservation of rat cardiomyocyte function using the common cardiac cross-bridge inhibitors 2,3-Butanedione monoxime (BDM), a phosphatase, and Blebbistatin, a selective myosin ATPase. We isolated cardiomyocytes from 6 mo old female Sprague Dawley rats. The isolate was divided for use over multiple days and storage conditions. Functional measurements (calcium transients and sarcomere length (SL) twitches) were obtained in a Tyrodes solution at 25°C beginning on the day of isolation (Day 0). Cells used on Days 1-4 post isolation were stored in a HEPES buffered solution with 0.5mM Ca⊥(2+) at 4°C: (1) without additional cross-bridge inhibitors (control), (2) +10mM BDM, and (3) +3µM Blebbistatin. Control (inhibition-free) cells showed reduced SL (<1.7µm diastolic), membrane blebbing, and altered functional measurements (calcium transients and SL twitches) within 24 hours. Cells stored in BDM maintained function for 24 hours but exhibited reduced diastolic SL and significant blebbing at 48 hours. Cells stored in blebbistatin showed excellent maintenance of diastolic SL and cell membrane quality more than 4 days post isolation. Calcium transients did not change after 4 days of storage in blebbistatin, but SL shortening was attenuated after 24 hrs. UV light exposure deactivated blebbistatin inhibition and restored SL shortening back to Day 0 magnitudes. Storage of rodent cardiomyocytes may help investigators reduce, replace, or refine experiments by allowing extended use of cardiomyocytes from single isolations.