Previously, we reported that distal Schaffer collaterals undergo biphasic changes in excitability during high-frequency stimulation (HFS), with an early hyper-excitability period followed by an excitability depression period. The extracellular divalent cations calcium and magnesium can regulate membrane excitability in neuronal tissue. Therefore, we hypothesized that altering the concentrations of extracellular calcium and magnesium would alter the biphasic excitability changes. We tested this hypothesis by recording distal Schaffer collateral fiber volleys in stratum radiatum of hippocampal area CA1 during 100 Hz HFS in artificial cerebral spinal fluid (ACSF) containing normal and altered concentrations of extracellular divalent cations. Our normal ACSF contained 2.0 mM calcium and 2.0 mM magnesium. We examined four solutions with altered divalent cation concentrations: (1) high-calcium/low-magnesium (3.8 mM/0.2 mM), (2) low-calcium/high-magnesium (0.2 mM/3.8 mM), (3) high-calcium/normal-magnesium (3.8 mM/2.0 mM), or (4) normal-calcium/high-magnesium (2.0 mM/10.0 mM), and assessed the effects on Schaffer collateral responses. Increasing or decreasing extracellular calcium enhanced or reduced (respectively) the early hyper-excitable period whereas increasing extracellular magnesium reduced the later excitability depression. Because these results might be explained by altered calcium influx through voltage-gated calcium (CaV) channels, we tested CaV blockers (ω-agatoxin IVA, ω-conotoxin-GVIA, cadmium), but observed no effects on responses during HFS. Some of the effects of altered divalent cation concentration may be explained by altered membrane surface charge. Although this mechanism does not completely explain our findings, calcium influx through CaV channels is not required.