Neuronal-glial interactions in the central nervous system are important for both normal function and response to pathological states. Differences in calcium processing between these cell types may be exploited to allow dynamic differentiation using calcium-imaging protocols without the need to fix and immunostain the study population. Mixed rat primary cortical cultures were grown on coverslips, incubated for 30 min in 2 microM fluo-3 AM and mounted in a devised, low volume imaging chamber. Calcium influx was measured over the duration of a 50s exposure to 30 microM glutamate in all cells. Cells were then fixed in situ, and immunostained for NeuN and GFAP. Direct comparison between live calcium dynamics and cell type markers were made. Over the duration of the glutamate exposure, those cells that subsequently stained for NeuN exhibited a sustained increase in intracellular calcium, whereas GFAP positive and non-staining cells exhibited a decline over the duration of the glutamate exposure. We found that examining the average calcium fluorescence over the last 10s of glutamate exposure allowed the identification of cells as neuronal if the average was >85% of the maximal calcium change, or non-neuronal if the average was <85% of the maximal calcium change. This technique compares very favourably to the established technique of immunocytochemical labeling for the identification of cell types; both techniques agreed in their classification of cells as neuronal or non-neuronal 96.83% of the time. However, this technique cannot reliably distinguish between non-neuronal cell types.