The [Ca²⁺]_i transient in heart is now thought to involve the recruitment and summation of discrete and independent “units” of Ca²⁺ release (Ca²⁺ “sparks”) from the sarcoplasmic reticulum, each of which is controlled locally by single coassociated L-type Ca²⁺ channels (“local control theory of excitation-contraction coupling”). All prior studies on Ca²⁺ sparks, however, have been performed in single enzymatically dissociated heart cells under nonphysiological conditions. In order to understand the possible significance of Ca²⁺ sparks to normal working cardiac muscle, we used confocal microscopy to record Ca²⁺ sparks, which are spatially averaged [Ca²⁺]_i transients (and Ca²⁺ waves), in individual cells of intact rat right ventricular trabeculae (composed of <15 cells in cross section) microinjected with the Ca²⁺ indicator fluo 3 under physiological conditions ([Ca²⁺]_o, 1 mmol/L; temperature, 33±1°C). Twitch force was recorded simultaneously. When stretched to optimal length (sarcomere length, 2.2 μm) and stimulated at 0.2 Hz, the trabeculae generated ≈700 μg of force per cell. Spatially averaged [Ca²⁺]_i transients recorded from individual cells within a trabecula were similar to those recorded previously from single cells. The amplitude distribution of the peak ratio of Ca²⁺ sparks was bimodal, with maxima at ratios of 1.8±0.3 and 2.7±0.2 (mean±SD), respectively. The amplitude of the peak of Ca²⁺ sparks was ≈170 nmol/L. Ca²⁺ sparks occurred at a frequency of 12.0±0.8/s (mean±SEM) in line scans covering 94 sarcomeres. Ca²⁺ waves occurred randomly at a frequency of 0.57±0.08/s and propagated with a velocity of 29.5±1.7 μm/s. The extent of Ca²⁺ wave propagation was 3.9±0.3 sarcomere lengths (sarcomere length, 2.2 μm). Ca²⁺ sparks could be identified along the leading edge of the waves at intervals of 1.30±0.11 sarcomere length. Our observations suggest that (1) Ca²⁺ sparks, similar to those recorded in single cells, occur in trabeculae under physiological conditions and (2) coupling of Ca²⁺ spark generation between neighboring sites occurs and may lead to (3) the development of Ca²⁺ waves, which propagate under physiological conditions at a low velocity over limited distances. The results suggest that concepts of excitation-contraction coupling recently derived from isolated myocytes are applicable to intact cardiac trabeculae.