A form of long-term potentiation (LTP) is induced at the mossy fiber (MF) synapse in the hippocampus by high-frequency presynaptic stimulation (HFS). It is generally accepted that induction of this form of LTP (MF LTP) does not depend on postsynaptic Ca2+ current gated by N-methyl-D-aspartate receptors, but it has remained controversial whether induction depends on postsynaptic depolarization and voltage-gated entry of Ca2+. There are also contradictory data on the time course of both LTP and post-tetanic potentiation (PTP), a shorter duration form of potentiation observed at MF synapses immediately following HFS. It has been proposed that some of these differences in results may have arisen because of difficulties in isolating monosynaptic responses to MF input. In the present study, whole cell recording was used to observe excitatory postsynaptic currents (EPSCs) elicited in CA3 pyramidal cells by input from MFs. Postsynaptic cells were dialyzed with 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and F- to inhibit postsynaptic mechanisms that required Ca2+, cells were under voltage clamp during HFS, and conditions were selected to minimize the likelihood of polysynaptic contamination. Under these conditions, HFS nevertheless induced robust LTP (mean magnitude, 62%). The possibility that EPSCs were contaminated by polysynaptic components was investigated by exposing the slices to a suppressing medium (one that partially blocked neurotransmission). EPSC waveforms did not change shape during suppression, indicating that contamination was absent. The LTP observed always was accompanied by prominent PTP that lasted through the first 5 to 15 min following HFS (mean decay time constant, 3.2 min). Induction of this LTP was not cooperative; there was no relationship between the size of responses and the magnitude of the LTP induced. LTP magnitude also was unrelated to the extent to which postsynaptic cells depolarized during HFS. These results show that high rates of presynaptic MF activity elicit robust LTP whether or not there is accompanying postsynaptic depolarization or increase in the concentration of postsynaptic Ca2+. High-frequency MF activity also results in a PTP that is unusually large and long.