The adenosine-producing ectoenzyme 5′-nucleotidase has recently been shown to undergo a marked redistribution during development of the cat visual cortex and to be involved in the remodelling of ocular dominance columns (Schoen et al., J. Comp. Neurol., 296, 379 – 392, 1990). Using an enzyme-cytochemical technique, we now investigate the developmental redistribution of 5′-nucleotidase activity in area 17 of kittens at the ultrastructural level. Between postnatal days 35 and 42, when 5′-nucleotidase is concentrated in layer IV, enzyme reaction product occupies the clefts of asymmetrical synapses within the neuropil. During later development (9th and 13th postnatal weeks), when 5′-nucleotidase spreads over all cortical laminae, the enzyme disappears from its synaptic localization and becomes increasingly associated with astrocytic membranes. The transient appearance of 5′-nucleotidase at synapses parallels the time-course and laminar profile of the synaptic remodelling which takes place during the critical period of visual cortex development. This suggests that synapse-bound 5′-nucleotidase activity plays a role in synaptic malleability, whereas its later association with glial profiles is likely to reflect other functions of the enzyme.