Cardiac fibrosis is a characteristic feature of left ventricular hypertrophy. The aim of this study was to develop a simple and accurate method to analyse collagen accumulation, taking into account the variation in cardiac muscle fibre orientation and nonuniform collagen distribution. This technique was used to determine the amount and types of collagen that accumulate during pressure overload cardiac hypertrophy. These data were correlated with myocyte size, and with the diastolic stress–strain relationship of the intact myocardium. Myocyte size was significantly increased in the hypertrophied hearts, compared with age and sex matched controls (control 363±25 μm2 vs experimental 244μm2; mean±S.E., P < 0.05). No overall collagen accumulation was observed in the hypertrophied hearts, but a significant increase in collagen I was found with a reduction in the amount of collagen III in experimental animals. Since no increase in diastolic stiffness of the hearts was observed, these results indicate that an increase in the overall collagen content of the heart, rather than the upregulation of a specific type, may be necessary to cause diastolic dysfunction.