The objectives of this study were to (1) determine whether the glycinergic and GABAergic amacrine cells in the developing rabbit retina were neurochemically distinct at birth, (2) determine if the ratio of GABAergic to glycinergic amacrine cells was constant during development, (3) determine whether the capacity to take up a GABA analogue was restricted to GABAergic neurons, and (4) whether initiation of GABA transport into GABAergic neurons preceded the presence of a content of GABA in these neurons. We have used a novel strategy to immunolocalize a non-endogenous GABA analogue, γ-vinyl GABA, which is taken up into neurons by a GABA transporter. Examination of serial semithin resin-embedded sections of neonatal rabbit retinae that had been immunolabelled for glycine, GABA or γ-vinyl GABA revealed that at 1 day postnatum, 60% of amacrine cells contain glycine but not GABA and did not accumulate γ-vinyl GABA, which is similar to the percentage of glycinergic amacrine cells in the adult retina. The vast majority of the remaining amacrine cells contained GABA and many also transported γ-vinyl GABA; however, a significant number of GABA-containing cells failed to accumulate γ-vinyl GABA suggesting that possession of a content of GABA did not have to be preceded by, or be concomitant with, the presence of a GABA transport system. By 10 days postnatum, over 99% of GABA-containing amacrine cells also transported γ-vinyl GABA indicating their functional maturity. Analysis of the horizontal cells revealed no evidence for uptake of γ-vinyl GABA, but another GABA analogue, diaminobutyric acid, which is a substrate both for the neuron-associated GABA transporter and the glial GABA transporter, was accumulated into some horizontal cells at 21 days postnatum, a time point when these cells also contain endogenous GABA. We conclude that amacrine cells are committed to being GABAergic or glycinergic at, or prior to birth, and that in some amacrine cells, expression of a content of GABA may occur prior to the capacity to transport GABA. Conversely, in some ganglion cells transport of γ-vinyl GABA may precede a content of GABA.