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In the CNS of leech, the central projections of peripheral sensory neurons segregate into three distinct axonal tracts during early development. We have previously shown that a subset of these neurons, recognized by the monoclonal antibody lan 4-2, projects axons into only one of these fascicles (Johansen et al., 1992, Neuron 8, 599). Here we report on a developmental and biochemical characterization of another fascicle-specific antigen labeled by the monoclonal antibody lan 3-6. By immunocytochemistry and double labelings we demonstrate that the lan 3-6 epitope is expressed only by a small subgroup of the peripheral neurons in Macrobdella embryos. The axons of these neurons selectively fasciculate in the CNS, but to only one of the three lan 3-2-positive tracts, which is different from the previously described lan 4-2-positive tract. These observations support the existence of a hierarchy of guidance cues mediating specific tract formation in this system. A biochemical analysis of the antigen suggests that it is likely to be a glycosylated protein with a molecular weight of approximately 200 kDa. Thus, the restricted expression of the lan 3-6 antigen and its biochemical properties are consistent with the hypothesis that this antigen may be playing a role in axonal guidance.