A mixed mesencephalic cell culture damaged by glutamate was used as a model to study the efflux of lactate dehydrogenase and neuron-specific enolase from neuronal cells into the culture medium. Glutamate toxicity was induced in sister cultures by 15 min exposure to 100 mumol/l glutamate in a Ca2+ containing salt solution. Cell injury was monitored 24 h later by measuring the lactate dehydrogenase activity and the neuron-specific enolase content in the cells and in the culture medium. The neuronal cell damage is reflected by an efflux of neuron-specific enolase and lactate dehydrogenase from the cells and an increase of lactate dehydrogenase catalytic activity concentration and neuron-specific enolase mass concentration in the culture medium. It was found that the efflux fraction calculated from estimations of the cells was clearly higher than the efflux fraction calculated from estimations of the amount of enzymes found in the culture medium. Calculations of the recovery of lactate dehydrogenase and neuron-specific enolase and experiments designed to study the efflux of lactate dehydrogenase and neuron-specific enolase during incubation and washing showed that higher amounts of neuron-specific enolase are released than lactate dehydrogenase. A close correlation was found between the glutamate-induced changes of the neuron-specific enolase efflux fraction, based on enzyme determinations of the cells, and the change of the microscopically counted neuron-specific enolase immunoreactive cell numbers. This indicates that the determination of the neuron-specific enolase efflux fraction (cells) is an accurate and sensitive marker of damaged neurons. The lactate dehydrogenase efflux fraction seems to be less sensitive for the quantitation of neuronal cell damage; in addition, it depends not only on the neuronal damage but also on the proportion of neurons in the cell culture.