The role of Ca in transmitter release and facilitation has been examined at the squid giant synapse by evaluating the effect of changes in the external concentration of Ca on the excitatory postsynaptic potential (EPSP). Changes in Ca were achieved by means of an arterial perfusion technique, circumventing the diffusion barrier that exists between the synapse and the bathing medium. Transmitter release was found to follow a high power (n) of the external Ca at low nonsaturating Ca concentrations and low stimulus frequencies: 4.0 +/- 0.1 (mean +/- SE) in 22 experiments. The value of n was not fixed, however, but declined at stimulus frequencies above 0.3 Hz to 2.9 at Hz, 2.3 at 10 Hz, 1.7 at 50 Hz, and 1.1 at 80 Hz. This decline in n was due to a greater proportional facilitation of the EPSP at lower Ca levels. In a separate experiment, facilitation was determined at a fixed low Ca level over a wide range of stimulus frequencies. A plot of the maximum percentage increase in the EPSP against the stimulus frequency described three "steps," with plateaus occurring at 0.3-10 Hz, 10-50 Hz, and over 80 Hz. The timing of these steps corresponds to the stimulus frequencies at which the cooperativity declines. The results of this study indicate that the cooperative action of four Ca ions are required to trigger the transmitter release mechanism, and that the phenomenon of facilitation involves a reduction in Ca cooperativity. It is proposed that Ca ions can bind up to four receptors on the release site and that binding results in the opening of "gates." The opening of all four gates triggers transmitter release.(ABSTRACT TRUNCATED AT 250 WORDS)