The prenatal and postnatal development of calretinin (CR)-containing elements in the neocortex of the rat was analyzed using immunohistochemistry. CR immunoreactivity in the cortical anlage appeared early at embryonic day 14 (E14), with CR-positive neurons located in the primitive plexiform layer and in the emerging subplate and marginal zones. At later prenatal and early postnatal stages, these two layers showed the highest CR immunostaining in the cortex, and large numbers of cell bodies and fibers were immunostained. From postnatal day 3 (P3) onwards, CR immunostaining disappeared progressively from the subplate-layer VIb and the marginal zone-layer I, so that very few cells remained stained in these layers in the adult. In the cortical plate and prospective layers VIa to II–III, CR-positive neurons were seen at prenatal stages, their numbers increasing markedly during the first postnatal week. Most neurons showed undifferentiated nonpyramidal shapes, and matured during the second and third postnatal weeks, when the adult pattern of CR immunostaining was achieved. In addition, some pyramidal-like neurons in the infragranular layers and in layer II–III transiently expressed CR during the postnatal period, most notably between P3 and P12. Colocalization experiments performed at P0–P3 with antibodies against the neurotransmitter γ-aminobutyricacid (GABA) showed that most nonpyramidal CR-positive neurons in the derivates of the cortical plate were also GABAergic during development. In contrast, large numbers of CR-containing neurons in the subplate and marginal zone were GABA-negative. The present results show that in addition to recording the early development of a subset of nonpyramidal neurons, CR is transiently expressed in certain GABA-negative populations of the subplate and marginal zone, and most likely in pyramidal neurons. © 1995 Wiley-Liss, Inc.