Chronic growth hormone (GH) hypersecretion in rats leads to increased isometric force without affecting the unloaded shortening velocity of isolated cardiac papillary muscles, despite a marked isomyosin shift toward V3. To determine if alterations occurred at the level of the contractile proteins in rats bearing a GH-secreting tumor (GH rats), we examined the mechanical properties of skinned fibers to eliminate the early steps of the excitation-contraction coupling mechanism. We found that maximal active tension and stiffness at saturating calcium concentrations (pCa 4.5) were markedly higher in GH rats than in control rats (tension, 52.9±5.2 versus 38.1±4.6 mN.mm−2,p<0.05; stiffness, 1,105±120 versus 685±88 mN · mm−2· μm−1,p<0.01), whereas values at low calcium concentrations (pCa 9) were unchanged. In addition, the calcium sensitivity of the contractile proteins was slightly but significantly higher in GH rats than in control rats (ΔpCa 0.04,p<0.001). The crossbridge cycling rate, reflected by the response to quick length changes, was lower in GH rats than in control rats (62.0±2.6 versus 77.4±6.6 sec−1,p<0.05), in good agreement with a decrease in the proportion of ce-myosin heavy chains in the corresponding papillary muscles (45.5±2.0% versus 94.6±2.4%,p<0.001). The changes in myosin heavy chain protein phenotype were paralleled by similar changes of the corresponding mRNAs, indicating that the latter occurred mainly at a pretranslational level. These results demonstrate that during chronic GH hypersecretion in rats, alterations at the myofibrillar level contribute to the increase in myocardial contractility observed in intact muscle.