Previous studies demonstrated increased fatty acid uptake and metabolism in MHC-FATP transgenic mice that overexpress fatty acid transport protein (FATP)1 in the heart under the control of the α-myosin heavy chain (α-MHC) promoter. Doppler tissue imaging and hemodynamic measurements revealed diastolic dysfunction, in the absence of changes in systolic function. The experiments here directly test the hypothesis that the diastolic dysfunction in MHC-FATP mice reflects impaired ventricular myocyte contractile function. In vitro imaging of isolated adult MHC-FATP ventricular myocytes revealed that mean diastolic sarcomere length is significantly (P<0.01) shorter than in wild-type (WT) cells (1.79±0.01 versus 1.84±0.01 μm). In addition, the relaxation rate (dL/dt) is significantly (P<0.05) slower in MHC-FATP than WT myocytes (1.58±0.09 versus 1.92±0.13 μm/s), whereas both fractional shortening and contraction rates are not different. Application of 40 mmol/L 2,3-butadionemonoxime (a nonspecific ATPase inhibitor that relaxes actin-myosin interactions) increased diastolic sarcomere length in both WT and MHC-FATP myocytes to the same length, suggesting that MHC-FATP myocytes are partially activated at rest. Direct measurements of intracellular Ca revealed that diastolic [Ca]i is unchanged in MHC-FATP myocytes and the rate of calcium removal is unexpectedly faster in MHC-FATP than WT myocytes. Moreover, diastolic sarcomere length in MHC-FATP and WT myocytes was unaffected by removal of extracellular Ca or by buffering of intracellular Ca with the Ca chelator BAPTA (100 μmol/L), indicating that elevated intracellular Ca does not underlie impaired diastolic function in MHC-FATP ventricular myocytes. Functional assessment of skinned myocytes, however, revealed that myofilament Ca sensitivity is markedly increased in MHC-FATP, compared with WT, ventricular cells. In addition, biochemical experiments demonstrated increased expression of the β-MHC isoform in MHC-FATP, compared with WT ventricles, which likely contributes to the slower relaxation rate observed in MHC-FATP myocytes. Collectively, these data demonstrate that derangements in lipid metabolism in MHC-FATP ventricles, which are similar to those observed in the diabetic heart, result in impaired diastolic function that primarily reflects changes in myofilament function, rather than altered Ca cycling.