We have characterized the dependence on membrane potential (V(m)) and calcium current (I(Ca)) of calcium-induced calcium release (CICR) from the junctional-SR (j-SR, in the subsarcolemmal (SS) space) and non-junctional-SR (nj-SR, in the central (CT) region of the cell) of cat atrial myocytes using whole-cell voltage-clamp together with spatially resolved laser-scanning confocal microscopy. Subsarcolemmal and central [Ca(2+)](i) transient amplitudes and I(Ca) had a bell-shaped dependence on V(m), but [Ca(2+)](i) reached a maximum at more negative V(m) (-10 to 0 mV) than I(Ca) (+10 mV). Termination of I(Ca) after a brief depolarization (2.5 to 22.5 ms) immediately interrupted only the SS [Ca(2+)](i) transient, leaving the development of the CT [Ca(2+)](i) transient unaffected. Block of SR function with 20 microM ryanodine and 2 microM thapsigargin, revealed that > 90 % of the control [Ca(2+)](i) transient amplitude was attributable to active SR Ca(2+) release through ryanodine receptors (RyRs). The gain of SR Ca(2+) release was highest in the SS space at negative test potentials and was less pronounced in the CT region. Inhibition of Na(+)-Ca(2+) exchange resulted in prolonged and higher amplitude [Ca(2+)](i) transients, elevated resting [Ca(2+)](i), accelerated propagation of CICR, decreased extrusion of Ca(2+) and an increase in j-SR Ca(2+) load. Increasing the cytosolic Ca(2+) buffer capacity by internal perfusion with 1 mM EGTA limited SR Ca(2+) release to the SS region, indicating that Ca(2+) release from nj-SR is initiated by diffusion of Ca(2+) from the cell periphery and propagating CICR. Junctional-SR Ca(2+) release occurred at discrete sites whose order of activation and amplitude of release varied from beat to beat. In conclusion, during normal excitation-contraction coupling in cat atrial myocytes, only Ca(2+) release from the j-SR is directly activated by Ca(2+) entering via I(Ca). Elevation of SS [Ca(2+)](i) is required to provide the cytosolic Ca(2+) gradient needed to initiate regenerative and propagating CICR from nj-SR.