During development of the central nervous system (CNS) the gene that encodes the 67 kDa form of glutamic acid decarboxylase (GAD) undergoes alternative splicing. The alternatively spliced variants include an exon (referred to as ES, for embryonic stop) that contains a premature stop codon. The detection of mRNA containing the ES exon in embryonic rat brain has been previously reported (Proc. Natl. Acad. Sci., 87 (1990) 8771-8775). We have used in situ hybridization to identify the anatomical distribution of ES mRNA in the embryonic rat brain during two stages of development, embryonic day 17 (E17) and E20. At E17, GAD67 mRNA was expressed in several CNS regions that were destined to contain GABAergic neurons when mature. ES transcripts were predominantly localized to ventricular zones and other regions associated with populations of proliferative cells at E17 and E20. At both ages, however, the alternatively spliced variants were also detected in regions of brain associated with migratory or post-mitotic neurons. GAD67 transcripts that did not include the ES exon were localized to anatomical areas that contained post-mitotic, and often post-migratory neurons. The temporal and spatial disappearance of mRNA containing the ES exon generally followed a caudal-to-rostral gradient which paralleled neuronal terminal mitosis and differentiation.