By using an antibody to glutaraldehyde fixation products of glycine we have been able to observe the development of a defined population of spinal interneurones in the CNS of Xenopus laevis embryos. The first glycine immunoreactive (GLY) somata appeared at stage 22 in the caudal hindbrain within a few hours of neural tube closure. The population then increased by extending caudally into the spinal cord and by infill. It was followed up to the time of hatching, stage 37/38. By observing GLY cells at early stages in their differentiation, the normal sequence of cell process formation was deduced. A ventral axon is formed, extends dendrites laterally into the marginal zone and forms a commissure by growing through the ventral ependymal cell floor of the neural tube. On the opposite side, growth cones turn longitudinally and TEM observations show that they make en-passant synaptic contacts. All GLY cells have decussating axons and some grow secondary axons on the same side as the soma. To establish the identity of GLY cells, a detailed comparison was made with commissural and dorsolateral commissural interneurones defined by retrograde and intracellular HRP staining. The GLY cells are identified with the commissural interneurones which are known to serve a glycinergic reciprocal inhibitory function. By showing that these interneurones have a clearly defined group identity and programme of development, this study opens the way to further experiments on factors controlling spinal cord pathway determination.