Neuronal nitric oxide synthase (nNOS) is important in cardiac protection in diseased heart. Recently, we have reported that nNOS is associated with myofilament Ca2+ desensitization in cardiac myocytes from hypertensive rats. So far, the effect of myofilament Ca2+ desensitization or nNOS on L-type Ca2+ channel activity (ICa) in cardiac myocyte is unclear. Here, we examined nNOS regulation of ICa in left ventricular (LV) myocytes from sham and angiotensin II (Ang II)-induced hypertensive rats.Our results showed that basal ICa was not different between sham and hypertension (from −60 to +40mV, 0.1Hz). S-methyl-l-thiocitrulline (SMTC), a selective nNOS inhibitor, increased peak ICa similarly in both groups. However, chelation of intracellular Ca2+ [Ca2+]i with BAPTA increased ICa and abolished SMTC-augmentation of ICa only in hypertension. Myofilament Ca2+ desensitization with butanedione monoxime (BDM), a myosin ATPase inhibitor, decreased ICa in both groups but to a greater extent in hypertension. Intracellular BAPTA or nNOS inhibition reinstated ICa in the presence of BDM to the basal level, suggesting Ca2+-dependent inactivation of ICa by nNOS and greater vulnerability in hypertension. Increasing stimulation frequencies (2, 4 and 8Hz) attenuated myofilament Ca2+ sensitivity in sham and reduced peak ICa in both groups. Nevertheless, SMTC or BAPTA exerted no effect on ICa at high frequencies in either group.These results suggest that nNOS attenuates ICavia Ca2+-dependent mechanism and the vulnerability is greater in hypertension subject to myofilament Ca2+ desensitization. nNOS or [Ca2+]i does not affect ICa at high stimulation frequencies. The results were recapitulated with computer simulation.