Agrin is a synaptic basal lamina protein that has been proposed to mediate motor neuron-induced clustering of acetylcholine receptors during development of the neuro-muscular junction. The chick ciliary ganglion is a parasympathetic ganglion that contains motor neurons that project to striated and smooth muscle targets in the eye. We have examined agrin gene expression in the chick ciliary ganglion during normal embryonic development using in situ hybridization and quantitative PCR techniques. Ganglia were specifically labeled by antisense agrin cRNA probes and the density of labeling changed during development. Hybridization was most intense in sections of ganglia obtained from embryos before embryonic Day 15 (E15), declining to relatively low levels by hatching at E20. Throughout embryonic development labeling was associated with glial cells, in addition to both ciliary and choroid neurons. Measurement of agrin mRNA levels by competitive PCR showed that agrin gene expression in the ganglion increased dramatically between E8 and E10, was sustained at high levels from E10 to E14, and declined thereafter. This time course is coincident with the period of synapse formation between ganglionic neurons and their peripheral targets. Previous studies in chick CNS have shown that alternative RNA splicing of a single exon encoding 11 amino acids gives rise to an active and an inactive agrin protein. Our analysis of RNA isolated from chick ciliary ganglia demonstrated that a second, previously uncharacterized exon encoding 8 amino acids can also be spliced into the same region. Alternative splicing of both the 8- and the 11-amino-acid exons results in expression of four distinct agrin transcripts in the ganglion. Changes in the level of total agrin mRNA in the ganglion reflect developmentally regulated changes in the levels of these alternatively spliced agrin isoforms. These results demonstrate that agrin is expressed in autonomic motor neurons of the peripheral nervous system and support a wider role for agrin as a synaptogenic protein, not limited to spinal motor neurons.