Serotonin is a native neuromodulator of synaptic transmission at glutamatergic neuromuscular junctions of crayfish limb muscles. During times of stress, serotonin binds to presynaptic receptors, which activate adenylyl cyclase to elevate presynaptic levels of cAMP. cAMP binds to two presynaptic target proteins, hyperpolarization and cyclic nucleotide-activated (HCN) ion channels and an exchange protein activated by cAMP (Epac), and activation of these effectors results in enhancement of transmitter release to action potentials. cAMP elevation also results in a small preterminal rise in [Ca(2+)](i), which we show here to result from Ca(2+) influx through the presynaptic HCN channels opened by cAMP. Little or no Ca(2+) influx occurs through voltage-dependent Ca(2+) channels, despite the small presynaptic depolarization caused by current through the HCN channels. Loading terminals with BAPTA delays the rise in preterminal [Ca(2+)](i) without affecting the enhancement of transmission to cAMP elevation. This dissociation of the dynamics of the [Ca(2+)](i) rise and synaptic enhancement, plus the small magnitude and location of [Ca(2+)](i) elevation distant from release sites, seems to preclude any direct role for this [Ca(2+)](i) elevation in cAMP-dependent enhancement of transmission.