The ontogeny of intersegmental (propriospinal) projections was studied in the chick embryo spinal cord between embryonic day 2.5 and day 6. Our goals were 1) to determine the earliest projections of intersegmental interneurons between specific spinal regions and to establish the cell types involved; and 2) to follow the ontogeny of these projections during the early formative stages of spinal cord development. Studies were carried out in vitro by using an isolated spinal cord/brainstem preparation. Horseradish peroxidase injections were made either uni- or bilaterally at various levels of the spinal cord along the rostrocaudal axis of the embryo. HRP histochemistry was done on Vibratome sections with diaminobenzidine as the chromogen. Following unilateral injections at day 2.5, labelled commissural interneurons were found contralaterally and were confined to the injected segment. Subsequently, labelled cells were found progressively further away from the injected segment. By day 4.5 reciprocal projections extended between lumbar and brachial regions. Interneurons with intersegmental axonal projections were often undifferentiated, consisting of primitive unipolar or bipolar cells with little, if any, dendritic development. In some cases migrating interneurons could be retrogradely labelled from two or three segments away from the location of their translocating cell body. Anterograde Golgi-like labelling of early undifferentiated cells revealed growing axons, axonal terminals, and growth cones. Five or six reasonably distinct classes of intersegmental interneurons were identified based on their location, axonal projections, and morphology of dendritic arbors. These appeared to be segmentally and bilaterally arranged along the rostrocaudal axis of the spinal cord. The axons of some of these types of interneurons exhibited preferences in their longitudinal projections within the ventral and ventrolateral marginal zone at the very onset of pathway formation. From the present observations it can be concluded that intersegmental connectivity precedes the development of ascending and descending supraspinal, as well as primary afferent connections in the chick embryo spinal cord.