Agrin is an extracellular matrix protein involved in clustering acetylcholine receptors during development of the neuromuscular junction. We have previously shown that alternative splicing at three sites generates multiple forms of rat agrin and that a novel 8 amino acid insert is the most important in determining biological activity. In the present study we have examined the expression of agrin during development with particular emphasis on determining the tissue distribution of the splicing variants at each site. Our principal observation is that the variants containing the sequence most responsible for biological activity are expressed exclusively in neural tissue and that their expression is highly regulated during development. We also show that muscle expresses less active forms and that agrin immunoreactivity during synaptogenesis is initially not limited to synaptic sites, but becomes progressively restricted to the synapse as development proceeds.