On the basis of cytochemical and morphologic differences, two classes of gamma-aminobutyric acidergic (GABAergic) interneurons expressing calcium-binding proteins have been identified in the striatum of adult animals: neurons expressing either parvalbumin (PV) or calretinin (CR). The function of these calcium-binding proteins is not clear, however, they are associated with distinct classes of inhibitory interneurons within the adult neostriatum. By using immunocytochemical techniques, we analyzed the postnatal maturation and the spatiotemporal distribution of PV- and CR-positive neurons in the rat neostriatum compared with a third class of interneurons characterized by the expression of the acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT). PV-positive cells appeared initially on postnatal day 9 in the lateral region of the striatum. During postnatal weeks 2 and 3, the numbers of PV-positive neurons increased, and this cell population spread progressively in a lateromedial direction. In contrast, CR-expressing neurons were present at birth. During the first few days after birth, the number of CR-immunoreactive cells increased, reaching a peak on postnatal day 5 before declining during the following 2 weeks. A mediolateral gradient was evident temporarily. ChAT-containing neurons were detectable at birth in the lateral striatum. During postnatal weeks 1 and 2, the neurons matured along a lateral-to-medial gradient. The results indicate that the maturation of striatal interneurons is regulated differentially during postnatal development, resulting in a distinct spatiotemporal genesis of phenotypes. The sequential expression of CR and PV suggests a stage-dependent development of subsets of inhibitory interneurons and, hence, the stage-dependent maturation of functionally distinct inhibitory circuits within the neostriatum.