As a means of defining functionally important regions of the L1 neuronal cell adhesion molecule, neurite outgrowth from cerebellar neurons was compared on monolayers of L1-negative B28 glioma cells, B28 cells transfected with wild-type human L1, and B28 cells transfected with variant forms of L1. Neurite outgrowth on L1-positive B28 cells is greatly enhanced over that seen on parental B28 cells. Neurite outgrowth on B28 cells expressing L1 variants that lack either the first or the fifth fibronectin type III repeat is comparable to that seen on monolayers expressing wild-type L1. In contrast, B28 cells expressing L1 without the third fibronectin type III repeat do not support neurite outgrowth above the background level seen on parental B28 cells. This suggests that the third fibronectin type III repeat plays a key role in the ability of L1 to promote neurite extension. This is consistent with reports that the third fibronectin type III repeat mediates L1 homomultimerization and integrin binding and that plasmin cleavage within this domain interferes with L1 function by abolishing these molecular interactions.