We systematically evaluated three different methods of preparing canine myocardium for NMR relaxometry: using whole specimens, specimens sliced into approximately 2-mm3 pieces, and specimens minced into pieces less than 2 mm3. NMR relaxation times were determined at 20 MHz. T1 relaxation time and water content were not different among the three methods. However, T2 values differed significantly between whole and sliced samples (mean +/- SEM = 51.2 +/- 2.2 ms (whole) vs 54.3 +/- 2.7 ms (sliced): P less than 0.05). Minced myocardium showed a similar increase that was not statistically significant. We conclude that tissue preparation methods must be controlled when performing NMR relaxometry studies.