Calcineurin is a ubiquitous calcium/calmodulin dependent protein phosphatase that has been shown to regulate the activity of ion channels, glutamate release, and synaptic plasticity. In the present study we show that CsA, a specific inhibitor of calcineurin, affects the survival of cultures developed from hippocampal dentate gyrus. Mixed neuronal-glial cultures exposed to 8 - 40 microM CsA undergo cell death characterized by apoptotic changes in cellular and nuclear morphology. TUNEL-positive staining was observed only in neurons that developed pyknotic morphology after treatment with 8 microM CsA for 24 - 72 h. Immunocytochemical staining with an anti-GFAP monoclonal antibody revealed that astrocytes from mixed neuronal/glial cultures were unaffected by exposure to CsA at doses toxic for neurons and all TUNEL-positive cells were neurons. MK-801, a noncompetitive inhibitor of glutamate receptor, does not inhibit the appearance of TUNEL-positive neurons and apoptotic changes in nuclear morphology. Preincubation of cells with 8 microM CsA increased basal intracellular calcium level in time dependent manner and decreased relative calcium response to glutamate. Application of 1 microM MK-801 had no effect on CsA-induced changes in Ca(2+) level. Our findings suggest that the neuronal death after CsA treatment is not a result of glutamate excitotoxicity and the increase in intracellular calcium concentration in neurons is not dependent on calcium influx via NMDA channel.