The ability to pre-label cells used in transplantation experiments would have the potential benefits of identification of cell type and associated processes and the analysis of graft migration in the host. We have used an in vitro tissue culture system as a model to test several fluorescent dyes for this application. Fetal rat hippocampal tissue (E17-E18) was dissociated and incubated in the presence of carboxyfluorescein ester (CFSE), rhodamine-B dextran amine (RBD), DiI, or rhodamine-labeled latex microspheres. Cells were cultured in defined medium for up to 1 month. Cells labeled with CFSE were initially bright but faded over several days. RBD labeled the soma of cells, but fluorescence intensity was lost over a period of a few weeks. Cells labeled with DiI possessed brilliant staining of neuronal processes for weeks. Latex microspheres brightly labeled the soma but not the processes of neurons; fluorescent debris and sterility were problems with this label. We conclude that CFSE and DiI have significant potential usefulness in vitro as markers of cell viability and process formation with mammalian fetal CNS cells, whereas RBD is much less permanent. Latex microspheres may be suitable for pre-labeling of cells for transplantation if purification and sterility can be enhanced over present preparations.