One major component of the extracellular matrix is hyaluronan (HA) which is thought to play a crucial role in the development of different organs including the central nervous system (CNS). HA is bound by specific receptors, CD44 and RHAMM, depending on cell types of CNS. However, data are lacking on the relation of HA to different cell populations in developing CNS. To provide new data about the co-localization of HA with the various cellular structures of the developing spinal cord, we studied the distribution pattern of hyaluronan in chicken embryos at Hamburger-Hamilton (HH) stages 8-39. A biotinylated HA-binding complex was used in combination with immunohistochemistry for proliferating and differentiating neurons. The intensity of the HA signal was determined by digital densitometry from histological sections. We found three mediolaterally oriented layers in the HA distribution pattern in stage HH23: (1) a moderate HA signal was detected in the ventricular zone; (2) strong HA accumulation was measured around Lim1,2-expressing cells (differentiating neurons) and early MNR2-expressing neurons (early motoneurons), corresponding to the intermediate zone; (3) a strong pericellular HA reaction was found around the neurons of the marginal zone. Interestingly, the peripheral nerves did not show HA signals. These findings suggest a crucial role of HA during neuronal development. We propose that HA may be involved in cell migration and axonal growth in the developing spinal cord.