The activities of choline acetyltransferase and glutamate decarboxylase were measured in individual layers of the adult and developing rat visual cortex. In the adult, the level of choline acetyltransferase activity was highest in layer V followed by layers I, II & III, IV and VI. These measurements are in complete agreement with recent immunohistochemical observations in the same cortical area. Glutamate decarboxylase activity was highest in layer IV and declined significantly in the more superficial and deeper layers. The activities of both enzymes were low during the first postnatal week but increased dramatically between days 8 and 18. Choline acetyltransferase activity in all layers demonstrated a more gradual rise to adult levels from day 18 onward, while glutamate decarboxylase activity reached adult levels by day 24 in all layers, except layer IV, which showed a continuous increase to adulthood. The functional role of the differences in the laminar distribution of these enzymes remains unknown.