There are two hypotheses to explain how neurons release transmitter. The calcium hypothesis proposes that membrane depolarization is necessary only for opening calcium channels and increasing internal calcium concentration ([Ca2+]i) near membrane transmitter-release sites. These calcium ions trigger a transient release of neurotransmitter. The calcium-voltage hypothesis postulates that voltage induces a conformational change in a membrane protein rendering it sensitive to calcium such that, in the presence of high [Ca2+]i, depolarization directly triggers transmitter release. Here we report that when calcium influx is blocked by cobalt or manganese ions in a calcium-free Ringer, as measured with Fura-2, and [Ca2+]i is elevated by liberation from a caged calcium compound, transmitter release at the crayfish neuromuscular junction is unaffected by presynaptic action potentials. These results support the calcium hypothesis.