SPINAL motoneurons are highly vulnerable to kainate bothin vivoandin vitro. Tissue-type plasminogen activator (tPA) and plasmin have recently been shown to mediate kainate-induced neuronal death in the mouse hippocampusin vivo. The aim of the present study was to determine whether tPA also mediates the kainate-induced death of motoneuronsin vitro. A motoneuron-enriched neuronal population was isolated from the ventral spinal cord of wild-type (WT) and tPA–deficient (tPA–/–) mouse embryos. WT and tPA–/–neurons were cultured on WT and tPA–/–spinal glial feeder layers, respectively. WT and tPA–/–co-cultures were morphologically indistinguishable. Expression of tPA in WT co-cultures was demonstrated using RT-PCR. WT and tPA–/–co-cultures were exposed to kainate for 24 h. The neurotoxic effect of kainate did not differ significantly between WT and tPA–/–cultures. The plasmin inhibitor α2-antiplasmin did not protect WT neurons against kainate-induced injury. These results indicate that the plasmin systemis not a universal mediator of kainate-induced excitotoxicity.