Agrin is an extracellular matrix protein that mediates the nerve- induced clustering of nicotinic acetylcholine receptors on target muscle cells, and thus plays a key role in development of the neuromuscular synapse. Alternative exon usage within the rat agrin gene predicts numerous protein isoforms, which differ by the inclusion or exclusion of small inserts at three sites in the C-terminal half of the molecule; the insert status at two of these sites, termed Y and Z, profoundly influences the acetylcholine receptor clustering activity. We have examined the cellular expression patterns of agrin messenger RNA transcripts during rat embryogenesis by in situ hybridization with isoform-specific probes. Six 36-mer oligonucleotide probes were designed to distinguish between mRNA isoforms at either the Y site: the encoded protein contains either no insert (Y0) or a 4-amino acid insert (Y4), or the Z site: the encoded protein contains either no insert or one of 8 (Z8), 11 (Z11), or 19 (Z19) amino acids. Strikingly different expression patterns were observed for the individual Y- and Z-site encoding messages. While optional exon usage predicts the possibility of eight different agrin isoforms at the two splice sites, we detected only four isoforms in vivo: Y4Z0, Y0Z0, Y4Z8, and Y4Z19. The Y4Z0 transcript, which comprised the majority of the agrin expressed, was localized exclusively to nervous tissue and exhibited a distribution profile suggestive of a potential role in neurogenesis and/or neural differentiation. From embryonic day 13 to birth, Y4Z0 was found in mitotic ventricular zones, spinal, cranial, and sympathetic ganglia, and diffusely throughout the brain. In contrast, Y0Z0 was not expressed in neurons, but specifically labeled capillary endothelial cells within the developing nervous system. Y4Z8 and Y4Z19, the forms most active in acetylcholine receptor aggregation, were expressed at low levels only in spinal and brainstem motor neurons; Z19 expression declined from embryonic day 15 to adulthood, whereas Z8 expression increased slightly during this period. Transcripts encoding the Z11 insert could not be detected. These data suggest potential novel biological roles for agrin beyond that originally proposed in synapse formation.