Agrin is a secreted glycoprotein with the ability to cluster cell surface molecules, including the nicotinic acetylcholine receptor (AchR) on muscle cells. Alternate splicing of agrin mRNA results in a family of agrin proteins which differ in their clustering potency. Neuronal-specific isoforms with the highest clustering activity play a role in clustering postsynaptic proteins at the neuromuscular junction. However, the function of agrin isoforms expressed in many nonneuronal tissues, and only weakly active in clustering assays, remains obscure. Monolayer cultures of Chinese hamster ovary (CHO) cells expressing a neuronal (agrin-19) or a nonneuronal (agrin-0) form of agrin were used to assay the effect of agrin on neurite outgrowth and cell attachment. These results were compared to outgrowth on control CHO cells expressing only drug resistance and on regions of CHO-agrin monolayers not expressing detectable levels of agrin. Neurite extension on confluent monolayers of agrin-0- or -19-expressing CHO cells was reduced substantially below that of controls. In one experiment neurite lengths were compared at 2 and 3 days after plating and suggested that neurite outgrowth may be stopped and not simply retarded. Attachment of sensory or motoneurons was nearly twofold higher to agrin monolayers than to control cells, showing that the inhibition is not a result of a nonpermissive environment. An agrin construct missing the C-terminal half, removing the major site of variability and clustering activity, was also tested. This construct did not reduce outgrowth, suggesting that the C-terminal half of the protein may be important in stopping growth as well as inducing clustering. These results expand the role of agrin in synaptogenesis as it may provide a stop signal at the myofiber surface and may anchor the presynaptic fibers to the eventual motor endplate .