The ability of Ca, Sr and Ba ions to support transmitter release was studied at the squid giant synapse by examining their respective actions on presynaptic current and post-synaptic responses. Transmitter-induced post-synaptic currents were smaller in Sr- than in Ca- containing solutions, and much smaller in Ba-containing solutions. The time course and amplitude of spontaneous miniature post-synaptic potentials were similar in the presence of all three divalent ions. Sr or Ba substitution has little effect on the resting potential of presynaptic terminals. In Sr-containing solutions, action potentials were similar in amplitude and time course to those recorded in Ca. Ba slightly prolonged action potential duration but had no effect on amplitude. Voltage-clamped presynaptic terminals exhibited inward Ca, Sr or Ba currents which were apparently carried through Ca channels. These currents were similar in amplitude and time course in all three ions, being somewhat larger in Ba. Although presynaptic currents were similar in these ions, transmitter release induced by these currents depended upon the divalent species entering the presynaptic terminal. Release was greatest in response to presynaptic current carried by Ca and smallest in response to current carried by Ba. Transfer curves relating presynaptic current to post-synaptic potential were sigmoidal in all three ions, and exhibited limiting slopes of approximately 2. Divalent cations differentially support transmitter release at the squid giant synapse in the sequence Ca greater than Sr much greater than Ba. The differential efficacy of the divalent cations is not due to post-synaptic alterations, presynaptic potential changes or differences in presynaptic divalent cation conductances. This sequence may reflect the cation selectivity of the exocytotic process responsible for transmitter release.