The neuron adhesion molecules NILE/L1 and NCAM may be involved in axonal guidance and cell recognition. To investigate all exposed membrane domains of single neurons, something which has not previously been done for any adhesion molecule, we used digitally processed scanning electron microscopy with a high-energy backscatter electron detector. This allowed a quantitative analysis of immunogold staining densities on all surfaces of isolated rat hippocampal neurons in culture to study NILE/L1 and NCAM expression independent of potentially inductive innervation. During early stages of neuritic extension, all growth cones showed similar NILE/L1 expression, but as soon as a single process extended farther than the others (by 20 hours), this putative axon and its growth cone generally showed a stronger level of NILE/L1 immunogold labeling than the other neurites. This is the earliest evidence of plasma membrane differentiation between axons and dendrites. With further neuritic growth, the relative NILE/L1 expression on axons and their growth cones continued to increase. In contrast to some earlier reports, NILE/L1 was expressed on axonal growth cones growing on both polylysine-coated glass and astrocyte substrates. Strong immunostaining for NCAM-related polysialic acid (PSA) was found on axonal growth cones and filopodia, suggesting that the homophilic adhesive action of NCAM may be reduced during axonal growth. PSA showed greater labeling on distal axons than on other areas of the neuron, indicating a variable NCAM-mediated adhesion on different regions of the same cell. Neither NILE/L1, NCAM, nor PSA appeared to show regional differences in axons fasciculating or defasciculating on themselves. A strong intercellular heterogeneity of NILE/L1, NCAM, and PSA expression levels on neurons in the same culture dish was found, suggesting that subsets of cells from the hippocampus may express biologically relevant differences in adhesion molecules compared to neighboring neurons. In light of the growing body of evidence pointing to the multifaceted array of homophilic and heterophilic binding interactions that NILE/L1 and NCAM may exhibit, and the functional importance of molecular densities, the quantitative data here support the hypothesis that sufficient cellular and subcellular heterogeneity exists for these molecules to be involved in some aspects of axonal guidance.