Incorporation of exogenously applied fluorescent lipids into living cells was exploited to probe cellular structure and function in living hippocampal and cerebellar slices as assessed by fluorescent imaging techniques and intracellular recording. Nitrobenzoxadiole-phosphatidylcholine (NBD-PC) and BODIPY phorbol ester, in vitro substrates of phospholipase activity and protein kinase C, respectively, were incorporated and distributed into specific cell populations. In the hippocampal slice, both probes labeled the somata and proximal dendrites of pyramidal and granule cells but were hetrogeneously distributed across the different hippocampal fields. Changes in fluorescent properties of NBD-PC in individual pyramidal cell and granule cell somata were quantified upon challenge with a muscarinic agonist known to modulate phospholipase A2 activity. In the cerebellar slice, both probes labeled Purkinje cell bodies and dendrites but only NBD-PC labeled stellate and granule cells. The cellular and functional specificity of these fluorescent lipid probes shows great promise for monitoring biochemical events in complex neuronal systems with significant spatial and temporal resolution.