The nonreceptor tyrosine protein kinases pp60c-src, p590fyn, and pp62c-yes are localized in growth cones of developing neurons, but their function is undefined. To determine whether these tyrosine kinases were capable of regulating substrate-dependent axon growth, cultures of cerebellar neurons from wild-type, src-, fyn-, and yes- mice were analyzed for neurite outgrowth on the neural cell adhesion molecule L1 or the extracellular matrix protein laminin. The rate of neurite extension on L1 was reduced in src-, but not in fyb- or yes- neurons. Neurite extension on laminin was unaltered in src-, fyn-, or yes- neurons, indicating that pp60c-src, p59fyn, or pp62c-yes is not likely to participate in integrin-dependent axon growth. These results demonstrate that pp60c-src is a component of the intracellular signaling pathway in L1-mediated axonal growth and suggest that Src-related nonreceptor tyrosine kinases may have distinct, non-redundant functions in the nervous system.