To study the effect of left ventricular (LV) hypertrophy on force and Ca²⁺handling in isolated rat myocardium, LV hypertrophy was induced in rats by banding of the abdominal aorta. After 16 wk, arterial pressure was assessed by catheterization. LV trabeculae were isolated and loaded with indo 1 salt by iontophoretic injection. Isometric force and intracellular free Ca²⁺concentration ([Ca²⁺]_i) were measured at stimulation frequencies between 0.25 and 3 Hz and rest intervals between 2 and 240 s. Sarcoplasmic reticulum (SR) Ca²⁺content was also investigated using rapid cooling contractures (RCC). Systolic and diastolic pressure as well as heart weight-to-body weight ratios were significantly elevated in banded compared with control animals (167 vs. 117 mmHg, 108 vs. 83 mmHg, and 4.6 vs. 4.0 mg/g, respectively). At high frequencies, twitch relaxation and [Ca²⁺]_idecline rates were significantly slower in banded compared with control rats, and diastolic [Ca²⁺]_iwas higher in the banded rat muscles (at 3 Hz, force half-time = 83 vs. 68 ms; time constant of [Ca²⁺]_idecline = 208 vs. 118 ms; and diastolic [Ca²⁺]_i= 505 vs. 353 nM). These differences could not be ascribed to altered Na⁺/Ca²⁺exchange, since twitch relaxation and Ca²⁺handling were not different between groups in the presence of caffeine (or cyclopiazonic acid plus ryanodine), where relaxation depends primarily on Na⁺/Ca²⁺exchange. After long rest intervals (≥120 s), control rats showed a significant rest potentiation of force and Ca²⁺transients, whereas banded rats did not. In addition, RCC amplitudes increased with rest in control but were unaltered in banded rats. In summary, pressure-overload hypertrophy was associated with slower twitch relaxation and [Ca²⁺]_idecline but also with blunted rest potentiation of twitches and SR Ca²⁺content of LV trabeculae. The decrease in SR Ca²⁺-ATPase function in banded rats may contribute to the observed diastolic dysfunction associated with pressure-overload hypertrophy.