Oxygen supply and demand of individual cardiomyocytes during the development of myocardial hypertrophy is studied using calibrated histochemical methods. An oxygen diffusion model is used to calculate the critical extracellular oxygen tension (PO_2,crit) required by cardiomyocytes to prevent hypoxia during hypertrophic growth, and determinants of PO_2,crit are estimated using calibrated histochemical methods for succinate dehydrogenase activity, cardiomyocyte cross‐sectional area, and myoglobin concentration. The model calculation demonstrates that it is essential to calibrate the histochemical methods, so that absolute values for the relevant parameters are obtained. The succinate dehydrogenase activity, which is proportional to the maximum rate of oxygen consumption, and the myoglobin concentration hardly change while the cardiomyocytes grow. The cross‐sectional area of the cardiomyocytes, which increases up to threefold in the right ventricular wall due to pulmonary hypertension in monocrotaline‐treated rats, is the most important determinant of PO_2,crit in this model of myocardial hypertrophy. The relationship between oxygen supply and demand at the level of the cardiomyocyte can be investigated using paired determinations of spatially integrated succinate dehydrogenase activity and capillary density. Hypoxia‐inducible factor 1α can be demonstrated by immunohistochemistry in cardiomyocytes with high PO_2,crit and increased spatially integrated succinate dehydrogenase activity, indicating that limited oxygen supply affects gene expression in these cells. We conclude that a mismatch of oxygen supply and demand may develop during hypertrophic growth, which can play a role in the transition from myocardial hypertrophy to heart failure. Microsc. Res. Tech. 58:412–420, 2002. © 2002 Wiley‐Liss, Inc.