The experimental procedures to simultaneously detect contractile activity and Ca(2+) transients by means of the Ca(2+) sensitive bioluminescent protein aequorin in multicellular preparations, and the fluorescent dye indo-1 in single myocytes, provide powerful tools to differentiate the regulatory mechanisms of intrinsic and external inotropic interventions in intact cardiac muscle. The regulatory process of cardiac excitation-contraction coupling is classified into three categories; upstream (Ca(2+) mobilization), central (Ca(2+) binding to troponin C), and/or downstream (thin filament regulation of troponin C property or crossbridge cycling and crossbridge cycling activity itself) mechanisms. While a marked increase in contractile activity by the Frank-Starling mechanism is associated with only a small alteration in Ca(2+) transients (downstream mechanism), the force-frequency relationship is primarily due to a frequency-dependent increase of Ca(2+) transients (upstream mechanism) in mammalian ventricular myocardium. The characteristics of regulation induced by beta- and alpha-adrenoceptor stimulation are very different between the two mechanisms: the former is associated with a pronounced facilitation of an upstream mechanism, whereas the latter is primarily due to modulation of central and/or downstream mechanisms. alpha-Adrenoceptor-mediated contractile regulation is mimicked by endothelin ET(A)- and angiotensin II AT(1)-receptor stimulation. Acidosis markedly suppresses the regulation induced by Ca(2+) mobilizers, but certain Ca(2+) sensitizers are able to induce the positive inotropic effect with central and/or downstream mechanisms even under pathophysiological conditions.