This study reports the spatio-temporal pattern of BEN expression (a molecule of the immunoglobulin superfamily) during early stages of the first axonal tract formation, in the fore- and midbrain of chick embryos [Hamburger and Hamilton (HH) stages 12-22]. The expression of BEN has been analysed using immunohistochemistry and non-radioactive in situ hybridization. Furthermore, double labelling experiments (combining anti-class III beta-tubulin, a pan-neuronal marker, and anti-BEN antibodies) have been carried out to determine whether BEN is expressed by all first axonal tracts. The first neurons expressing BEN appear around stage HH13-14, in the caudal diencephalon. They belong to the interstitial nucleus of Cajal, and their axons are the first components of the medial longitudinal fasciculus. By HH14, two other early axonal tracts appear: the tract of the postoptic commissure and the descending root of the mesencephalic nucleus of the trigeminal nerve. Only the latter expresses BEN. At later stages of development numerous new axonal tracts appear in the telencephalic, diencephalic and mesencephalic domains. Only a few of them (the fourth nerve, the lemniscus lateralis, the tectobulbar and habenulopeduncular tracts) express BEN. In all BEN positive systems, the cell bodies, axons and growth cones are uniformly labelled by the antibody. We have found that none of the early axonal tracts grows preferentially at interneuromeric boundaries. Moreover, each tract is formed by several thin fascicles rather than a single one. The expression of BEN is transient and disappears shortly before hatching. These results suggest that BEN may serve to promote axonal outgrowth of precise neuronal systems involved in 'axonal scaffolding'.