Four isoforms of cardiac troponin T (cTnT), a protein essential for calcium-dependent myocardial force development, are expressed in the human; they differ in charge and length. Their expression is regulated developmentally and is affected by disease states. Human cTnT isoform effects have been examined in reconstituted myofilaments. In this study, we evaluated the modulatory effects of overexpressing one cTnT isoform on in vitro and in vivo myocardial function. A human cTnT isoform, hcTnT₁, expressed during development and in heart disease but not in the normal adult heart, was expressed in transgenic (TG) mice (1-30% of total cTnT). Maximal active tension measured in skinned myocardium decreased as a function of relative hcTnT₁ expression. The pCa at half-maximal force development (pCa₅₀), Hill coefficient, and rate of redevelopment of force did not change significantly with hcTnT₁ expression. In vivo maximum rates of rise and fall of left ventricular pressure decreased, and the half-time of isovolumic relaxation increased, with hcTnT₁ expression. Substituting total cTnT charge for hcTnT₁ expression resulted in similar conclusions. Morphometric analysis and electron microscopy revealed no differences between wild type (NTG) and transgenic (TG) myocardium. No differences in isoform expression of tropomyosin, myosin heavy chain, essential and regulatory myosin light chains (MLC), TnI, or in post-translational modifications of mouse cTnT, cTnI or regulatory MLC were observed. These results support the hypothesis that cTnT isoform amino-terminal differences affect myofilament function and suggest that hcTnT₁ expression levels present during human development and in human heart disease can affect in vivo ventricular function.