We determined whether two classes of Drosophila larval motor terminals with known differences in structure and transmitter release also showed differences in Ca(2+) regulation. Larval motor neurons can be separated into those producing large synaptic boutons (Ib) and those with small boutons (Is). Ib terminals release less transmitter during single action potentials (APs) than Is terminals, but show greater facilitation during high-frequency stimulation. We measured Ca(2+) transients produced by single APs and AP trains after loading the terminals with the dextran-conjugated Ca(2+) indicator Oregon Green 488 BAPTA-1 (OGB-1). The two pairs of Is and Ib terminals innervating muscle fiber 4 and fibers 6 and 7 were examined. The OGB-1 concentrations were measured in order to compare measurements from terminals with similar OGB-1 loading. For single APs, the change in OGB-1 fluorescence (DeltaF/F) in Is boutons was significantly larger than in Ib boutons due to greater Ca(2+) influx per bouton volume. The Is boutons had greater surface area and active zone number per bouton volume than Ib boutons; this could account for the differences in Ca(2+) influx and argues for similar Ca(2+) influx at Is and Ib active zones. As previously reported for the Ib boutons, the distal Is boutons had larger single-AP Ca(2+) transients than proximal ones on muscle fibers 6 and 7, but not on fiber 4. This difference was not due to proximal-distal differences in surface area or active zones per bouton volume and may be due to greater Ca(2+) influx at distal active zones. During AP trains, the Is Ca(2+) transients were larger in amplitude and had longer decay time constants than Ib ones. This can be explained by a slower rate of Ca(2+) extrusion from the Is boutons apparently due to lower plasma membrane Ca(2+) ATPase activity at Is boutons compared to Ib boutons.