Physical inactivity is an independent risk factor for coronary heart disease, yet the mechanism(s) of exercise-related cardioprotection remains unknown. We tested the hypothesis that coronary smooth muscle after exercise training would have decreased mitogen-induced phenotypic modulation and enhanced regulation of nuclear Ca²⁺. Yucatan swine were endurance exercise trained (EX) on a treadmill for 16-20 wk. EX reduced endothelin-1-induced DNA content by 40% compared with sedentary (SED) swine (P < 0.01). EX decreased single cell peak endothelin-1-induced cytosolic Ca²⁺ responses compared with SED by 16% and peak nuclear Ca²⁺ responses by 33% (P < 0.05), as determined by confocal microscopy. On the basis of these results, we hypothesized that sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and intracellular Ca²⁺ stores in native smooth muscle are spatially localized to dissociate cytosolic Ca²⁺ and nuclear Ca²⁺. Subcellular localization of SERCA in living and fixed cells revealed a distribution of SERCA near the sarcolemma and on the nuclear envelope. These results show that EX enhances nuclear Ca²⁺ regulation, possibly via SERCA, which may be one mechanism by which coronary smooth muscle cells from EX are less responsive to mitogen-induced phenotypic modulation.