The plasma membrane Ca²⁺ ATPase (PMCA) plays a major role in restoring Ca²⁺ to basal levels following transient elevation by neuronal activity. Here, we examined the effects of various stimuli that increase [Ca²⁺]_i on PMCA-mediated Ca²⁺ clearance from hippocampal neurons. We used indo-1 based microfluorimetry in the presence of cyclopiazonic acid to study the rate of PMCA-mediated recovery of Ca²⁺ elevated by a brief train of action potentials. [Ca²⁺]_i recovery was described by an exponential decay and the time constant provided an index of PMCA-mediated Ca²⁺ clearance. PMCA function was assessed before and for at least 60 min following a 10 minute priming stimulus of either 100 µM NMDA, 0.1 mM Mg²⁺ (reduced extracellular Mg²⁺ induces intense excitatory synaptic activity), 30 mM K⁺, or control buffer. Recovery kinetics slowed progressively following priming with NMDA or 0.1 mM Mg²⁺; in contrast, Ca²⁺ clearance initially accelerated and then slowly returned to initial rates following priming with 30 mM K⁺-induced depolarization. Treatment with 10 µM calpeptin, an inhibitor of the Ca²⁺ activated protease calpain, prevented the slowing of kinetics observed following treatment with NMDA, but had no affect on the recovery kinetics of control cells. Calpeptin also blocked the rapid acceleration of Ca²⁺ clearance following depolarization. In calpeptin-treated cells, 0.1 mM Mg²⁺ induced a graded acceleration of Ca²⁺ clearance. Thus, in spite of producing comparable increases in [Ca²⁺]_i, activation of NMDA receptors, depolarization-induced activation of voltage-gated Ca²⁺ channels and excitatory synaptic activity each uniquely affected Ca²⁺ clearance kinetics mediated by the PMCA.