An in vivo method for positively staining dead neurons was developed and compared with an in vitro staining method using acid fuchsin. Neurons previously killed by intracerebral injections of kainic acid were selectively stained by trypan blue within 15 min of its injection in vivo into the central nervous system of rats. Such staining persisted for at least 4 days in vivo, and there was no evidence that trypan blue itself was toxic to the remaining cells. Intense staining of neurons with acid fuchsin in vitro was first observed in brain sections of rats killed about 6h after kainic acid injection. This time was approximately 2-3 h before trypan blue, in vivo, was able to stain neurons. Thus, the loss of transport mechanisms (at least for trypan blue) apparently occurs subsequent to the development of basic products stainable with acidic dyes. At the earliest times, acid fuchsin stained neurons which had not yet lost Nissl substance, whereas the majority of trypan blue-stained neurons were not stained with Nissl dyes. After 24 h the majority of neurons stained with either acid fuchsin or trypan blue were Nissl-stain negative. The combination of staining with trypan blue in vivo with subsequent counterstaining of brain sections with acid fuchsin in vitro may have a potential use in the determination of the time of neuronal death in vivo.