The role of protein phosphatases in regulating synaptic transmission in the CA1 region of the hippocampus was examined using slices pretreated with calyculin A, a specific inhibitor of protein phosphatases 1 and 2A. Stimulation of afferents at 1 Hz (but not 0.1 Hz) for periods of 5– 10 min caused a long-lasting enhancement of synaptic transmission. The increase in synaptic responses was not due to a change in fiber excitability, as there was a shift to the left in the input-output curve following the synaptic enhancement. The enhancement was observed only in the input that received the 1 Hz stimulation and not in an independent control pathway, indicating that the increase in synaptic strength is input specific and limited to repetitively activated synapses. Applying 1 Hz stimulation when synaptic transmission was blocked by replacing extracellular Ca2+ with Mg2+ prevented or significantly reduced any change in synaptic efficacy after reperfusion with normal Ca(2+)-containing medium. In contrast, 1 Hz stimulation given when synaptic transmission was blocked by non-NMDA and NMDA glutamate receptor antagonists still caused a synaptic enhancement following washout of the antagonists. The enhancement of synaptic transmission also was not blocked by loading CA1 cells with the calcium chelator BAPTA. Thus, influx of Ca2+ into presynaptic elements is required for the synaptic enhancement elicited by 1 Hz stimulation in calyculin A-treated hippocampal slices. Consistent with the activation of processes that cause an increase in transmitter release, the magnitude of paired-pulse facilitation decreased following the synaptic enhancement, and the NMDA receptor-mediated component of the synaptic response was increased by 1 Hz stimulation. These results suggest that when protein phosphatases are inhibited by calyculin A, prolonged periods of 1 Hz stimulation lead to activation of presynaptic Ca(2+)- dependent protein kinases, resulting in a persistent increase in evoked transmitter release. They also indicate that the activity of presynaptic protein phosphatases is critically important for limiting increases in synaptic strength following repetitive afferent activity.