The distal Ca²⁺-binding domain of Synaptotagmin I (Syt I), C2B, has two Ca²⁺-binding sites. To study their function in Drosophila, pairs of aspartates were mutated to asparagines, and the mutated syt I was expressed in the syt I-null background (P[syt I^B-D1,2N] and P[syt I^B-D3,4N]). We examined the effects of these mutations on nerve-evoked synchronous synaptic transmission and high K⁺-induced quantal events at embryonic neuromuscular junctions. The P[syt I^B-D1,2N] mutation virtually abolished synaptic transmission, while the P[syt I^B-D3,4N] mutation strongly reduced but did not abolish it. The quantal content in P[syt I^B-D3,4N] increased with the external Ca²⁺ concentration, [Ca²⁺]_e, with a slope of 1.86 in double logarithmic plot, while that of control was 2.88. In high K⁺ solutions the quantal event frequency in P[syt I^B-D3,4N] increased progressively with [Ca²⁺]_e between 0 and 0.15 mM as in control. In contrast, in P[syt I^B-D1,2N] the event frequency did not increase progressively between 0 and 0.15 mM and was significantly lower at 0.15 mM [Ca²⁺]_e than at 0.05 mM. The P[syt I^B-D1,2N] mutation inhibits high K⁺-induced quantal release in a narrow range of [Ca²⁺]_e (negative regulatory function). When Sr²⁺ substituted for Ca²⁺, nerve-evoked synchronous synaptic transmission was severely depressed, and delayed asynchronous release was markedly increased in control embryos. In high K⁺ solutions with Sr²⁺, the quantal event frequency was higher than in Ca²⁺ and increased progressively with [Sr²⁺]_e in control and in both mutants. Sr²⁺ partially substitutes for Ca²⁺ in synchronous release but does not support the negative regulatory function of Syt I.