The expression of the homeobox-containing gene En-2 was analysed with the monoclonal antibody 4D9 in the chick central nervous system throughout embryogenesis. Confirming previous studies, early expression of the En-2 protein [beginning at stage 9 of Hamburger and Hamilton (HH9)] is restricted to a portion of the neural tube containing the primordia of the cerebellum, the isthmic region and the mesencephalic grisea, and forms a double gradient decreasing both caudally and rostrally from a high point located around the midbrain-hindbrain constriction. This mes-isthmo-cerebellar region contains all the En-2-positive germinative cells and the great majority of the En-2-positive postmitotic neurons throughout embryogenesis. Nevertheless, as the postmitotic neurons appear, En-2 expression also occurs outside this region: in two columns of non-motoneuron cells in rhombomeres two to four (between HH20 and HH30) and, from HH24 onwards, throughout the grey matter of the lumbar and thoracic spinal cord, with the exception of the ventral motoneuron columns. Here, a detailed description of En-2 expression is provided for the mes-isthmo-cerebellar region at stages HH30–32 [embryonic day (E) 71, HH37 (E11) and HH46 (E21, hatching). This allows the visualization of cellular groups with heterogeneous patterns of En-2 expression, which are specific for each group in the intensity of En-2 expression, the distribution of the labelled cells and the temporal regulation of the gene. The use of tyrosine hydroxylase antiserum shows coexpression of the tyrosine hydroxylase enzyme and En-2 protein in the caudal part of the nuclei tegmenti pedunculo-pontinus, the area ventralis of Tsai and the substantia grisea centralis, but not in the locus coeruleus. In the cerebellum, the first expression, which is located in the deep nuclei and parasagittal bands of Purkinje cells, is down-regulated when the molecular layer interneurons and the granular cells begin to express the gene, at the end of embryogenesis. Finally, at hatching, En-2 expression permits the visualization in the cerebellum of a population of small En-2-negative cells located around the Purkinje cells that may correspond to those described in chick/quail chimaeras as having an origin different from that of the bulk of granular neurons.