In order to learn about the factors regulating the postnatal development of neocortical peptidergic neuron populations, we have analysed neurons expressing neuropeptide Y (NPY) by immunohistochemistry and in situ hybridization in developing and adult rat visual cortical areas 17 and 18a in vivo, and in organotypic slice cultures of rat visual cortex. For quantitative analysis, the percentage of NPY mRNA-expressing neurons was determined in supragranular layers I–IV, in infragranular layers V and VI and in the white matter. In vivo, this percentage increased in visual areas 17 and 18a until postnatal day 21 in supra- and infragranular layers. Initially, in both areas the neurons were about equally distributed in supra- and infragranular layers (a ratio of 1:1). During the second postnatal month, the percentage of NPY mRNA-expressing neurons in area 18a declined by ∼50% in both supra- and infragranular layers, so that the ratio of 1:1 remained constant. In contrast, in area 17 the percentage of neurons in supragranular layers remained fairly constant, but it declined to 50% in infragranular layers, so that by postnatal day 70 the ratio was gradually shifted to 2:1. Throughout development, area 18a contained significantly more NPY mRNA-expressing neurons than area 17. In organotypic slice cultures, a high density of NPY mRNA-expressing neurons had appeared by 10 days in vitro. A much higher percentage of neurons expressed NPY mRNA. The ratio of labelled neurons in supra- versus infragranular layers was 1:1. Both ratio and percentage remained constant from 10–85 days in vitro. The decline in vivo was not caused by an elimination of transient cell types. All cell types persisted into adulthood. Four NPY peptide-immunoreactive neuronal types were classified by axonal morphology in organotypic slice cultures and in vivo; they include (i) cells in layer VI/white matter with horizontal axons and ascending collaterals, (ii) cells in layers V/VI with descending axon and horizontal collaterals, (iii) Martinotti cells in layers VA/I with ascending axons, and (iv) cells in layers III-V with columnar axons. Two further types, bipolar cells with axons descending from dendrites and small basket cells with short horizontal axons, both found in vivo in layers II/III, could not be unequivocally identified in organotypic slice cultures. The NPY-immunoreactive neuron types had already formed a dense innervation of the cultures by 10 days in vitro, which remained stable for up to 85 days in vitro, and resembled the innervation observed in vivo. NPY peptide-immunoreactive neurons in organotypic slice cultures and in vivo were distributed in cortical layers II/III, V and VI and the white matter, but rarely in layers I and IV, which corresponded to the distribution of NPY mRNA-expressing neurons. However, with in situ hybridization more neurons were detectable, especially in layers II/III. A majority of NPY mRNA-expressing neurons co-localized NPY peptide, somatostatin and calbindin. We conclude that intrinsic cues were sufficient to drive the molecular expression of the NPY phenotype, the morphological differentiation and the stabilization of an organotypic NPY innervation in organotypic slice cultures. However, the area- and lamina-specific changes observed in vivo were not observed under monoculture conditions.