Serotonin (5-HT) has multiple physiological actions at lobster neuromuscular junctions, including facilitation of transmitter release from nerve terminals and an increase in the tone and excitability of muscle fibers. These physiological effects of 5-HT are accompanied by a rise in intracellular levels of cAMP. We have used combined biochemical and physiological approaches to investigate whether cAMP directly mediates the physiological actions of the hormone. Based on the following lines of evidence, we conclude that the postsynaptic increase in muscle tone occurs independently of cAMP and that while the cyclic nucleotide does play a role in the facilitation of transmitter release by 5-HT, there is also a cAMP-independent component to this facilitation. (1) Agents that mimic the action of 5-HT on cAMP levels (forskolin, IBMX, SQ20009, 8-bromo cAMP) fail to mimic the postsynaptic actions of the amine. These agents do facilitate transmitter release, although none of them has as large an effect as does 5-HT. (2) When 5-HT is removed, presynaptic facilitation decays as the sum of 2 exponentials with very different time courses. The rate of the slower process is similar to the rate of breakdown of cAMP, while the faster process and the postsynaptic response decay significantly more rapidly. (3) IBMX retards the breakdown of cAMP and simultaneously retards the decay of the slower presynaptic process, with little or no effect on the other responses. (4) IBMX and forskolin potentiate the effect of 5-HT on cAMP levels and selectively enhance the slowly decaying presynaptic component with little or no effect on the other responses.