The effect of passive muscle stretch on the extent of MLC2v phosphorylation was investigated. We used an isolated rat heart preparation and controlled the passive pressure of the left ventricle (LV) at 0 or 15mmHg. The hearts were flash frozen and the LV free wall was split into epicardial and the endocardial halves. The samples were solubilized using a novel method that minimizes changes in the phosphate content of MLC2v under non-denaturing conditions. The proteins were separated by urea glycerol PAGE and identified by mass spectrometry and Western blots. At 0mmHg passive pressure, the extent of MLC2v phosphorylation of the epicardium (34.1±1.7%) was the same as that of the endocardium (35.3±3.4%). At 15mmHg passive pressure, we found a significant increase in MLC2v phosphorylation in the epicardium (to 41.5±2.0%) and a significant reduction in the endocardium (to 24.2±1.2%), giving rise to a gradient in the extent of MLC2v phosphorylation from epicardium (high) to endocardium (low). These changes in MLC2v phosphorylation that take place in response to increased diastolic pressure are likely to impact on the calcium sensitivity of actomyosin interaction (with an increased sensitivity towards the epicardium) and may play a role in the Frank–Starling mechanism of the heart.