We developed and characterized an assay that allows for rapid examination of migration of specific neuronal populations within a mixed population using the Boyden chamber principle. Migration of cerebellar interneurons and granule cells was examined using mice expressing enhanced green fluorescent protein (eGFP) under the glutamate decarboxylase (GAD(65)) and growth-associated protein-43 (GAP43) promoters, respectively. Brain-derived neurotrophic factor (BDNF) was used as the prototypic motogen for both populations. Fluorescent light-blocking inserts (FluoroBlok) with different pore sizes and densities were compared in a two-compartment assay. Immunodetection of polarity markers and nuclear staining indicated that dendrites and somata are preferentially extended through the pores in response to BDNF. Inserts coated with extracellular matrix (ECM) proteins were used to examine interactions between BDNF and the ECM during migration. ECM proteins alone stimulated migration when the lower side of the insert was coated, however coating of both sides of the insert slowed migration when compared to poly-D-lysine. Addition of a PI 3-kinase inhibitor to the lower compartment blocked BDNF-stimulated migration of both populations while a Src inhibitor reduced laminin-stimulated migration of interneurons, but not granule cells. We also examined use of neurons cultured from GAD(65)-eGFP mice as a reporter system for promoter activity. GAD(65)-eGFP mice may also be useful as a model for promoter regulation and the potential confounding effects of eGFP induction by the stimuli are also addressed. This assay allows for rapid analysis of motogens, substrates and signaling pathways that regulate migration of selected neuronal populations.