The extracellular matrix molecules tenascin, laminin, and fibronectin, the cell adhesion molecule L1, and the lectin concanavalin A (ConA) were tested for their effects on neuritogenesis in cultures of hippocampal neurons. We analyzed neurite outgrowth between 3 and 21 hr after plating and found that, on polyornithine as control substrate, lengths of axon-like major neurites and dendrite-like minor neurites increased continuously with time in culture. Moreover, growth of minor neurites was faster than growth of major neurites. When the extracellular matrix molecules tenascin, laminin, and fibronectin were coated on polyornithine substrates, growth of all neurites was faster than on control substrates during the first hours of culture. After this initial phase of enhanced neurite outgrowth, elongation of major neurites continued at a higher rate than on the control substrate and growth of minor neurites ceased after 12 hr. Correspondingly, neuronal polarity was strongly increased on the extracellular matrix substrates during later phases of culture. In contrast, lengths of both major and minor neurites were increased over control values on L1 and ConA substrates at all time points investigated. Thus, neuronal polarity was similar for control, L1, and ConA substrates. Spreading of neuronal cell bodies was reduced by about 50% on tenascin, laminin, and fibronectin and by less than 20% on L1 and ConA substrates after 21 hr of culture, when compared to the control substrate. Neuron-to-substrate adhesion was reduced on all three extracellular matrix substrates but not affected on L1 or ConA substrates, after 3 and 21 hr of culture. These observations indicate that induction of neuronal polarity is not a general feature of neurite outgrowth-promoting molecules, such as L1 or ConA, but a distinctive property of the three extracellular matrix glycoproteins studied, and may suggest that enhancement of polarity is correlated with decreased strength of adhesion.