α1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+sensitivity and action potential prolongation, which can increase net Ca2+entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2–0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+entry and differentiate between the two mechanisms. At 22–23°C the specific α1-adrenoceptor agonist methoxamine (100 μM) prolonged action potential duration at 50% repolarization from 55±2 to 81±5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3±0.6 to 7.8±1 μm (n=18). Reduction of the transient outward current and other K+currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52±6 to 98±12 msec and increased unloaded cell shortening from 2.9±0.4 to 6.6±0.6 p.m (n=4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70–500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3±0.7 to 4.97±0.8 μm at pulse potentials positive to −40 mV. Similar al-adrenoceptor responses were observed at 35°C during action potential and voltage-clamp experiments, which could be blocked by 10 μM prazosin. In myocytes loaded with the Ca2+indicator indo-1, α1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to α1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+current reduction. In addition, modulation of K+channels could occur independent of changes in contractility and/or [Ca2+];.