Synaptic functional differentiation of crayfish phasic and tonic motor neurons is large. For one impulse, quantal release of neurotransmitter is typically 100-1000 times higher for phasic synapses. We tested the hypothesis that differences in synaptic strength are determined by differences in synaptic calcium entry. Calcium signals were measured with the injected calcium indicator dyes Calcium Green-1 and fura-2. Estimated Ca(2+) entry increased almost linearly with frequency for both axons and was two to three times larger in phasic terminals. Tonic terminal Ca(2+) at 10 Hz exceeded phasic terminal Ca(2+) at 1 Hz, yet transmitter release was much higher for phasic terminals at these frequencies. Freeze-fracture images of synapses revealed on average similar numbers of prominent presynaptic active zone particles (putative ion channels) for both neurons and a two- to fourfold phasic/tonic ratio of active zones per terminal volume. This can account for the larger calcium signals seen in phasic terminals. Thus, differences in synaptic strength are less closely linked to differences in synaptic channel properties and calcium entry than to differences in calcium sensitivity of transmitter release.