Peripheral-type benzodiazepine receptors (PBRs) in brain are primarily localized within astroglial cells, and the existence of PBR subtypes have been suggested. In the present study the ontogeny of PBRs labeled with [3H]Ro5-4864 and [3H]PK 11195 in cultured astrocytes was compared to their postnatal in-vivo development. The density of [3H]Ro5-4864 binding sites in cultured astrocytes from rat cortex progressively increased from 1- to 3-week-old cultures and did not change further in 5- and 8-week-old cultures. The density of [3H]PK 11195 binding sites progressively increased from 1- to 5-week-old cultures. The density of [3H]PK 11195 binding sites exceeded the density of [3H]Ro5-4864 binding sites by 40-50% in 2-, 5- and 8-week-old cultures. The affinity of the PBR ligands for the receptor sites was increased by 3- to 4-fold from the first to the second week in cultures, and did not change thereafter. A similar developmental pattern of PBRs was observed in rat cortex, except that: first, the difference between the Bmax of [3H]PK 11195 and [3H]Ro5-4864 was already apparent in postnatal-week-1 and persisted with maturation; second, the high affinity of the ligands for the receptor sites was apparent from postnatal-week-1 and did not change with maturation. Age-related differences in the ratio between the density of PBRs in astrocytes and rat cortex were also observed. These results lead us to suggest that the development of PBRs in vivo during the first postnatal week is more rapid than the development of the receptors in vitro during the first week in culture. Subsequently, the increased ratio between the density of PBRs in cultured astrocytes and brain with maturation indicates the predominantly astrocytic localization of these receptors. The finding that the density of [3H]PK 11195 binding sites in cultured astrocytes and in rat brain cortex is usually 40-50% greater than the density of [3H]Ro5-4864 binding sites further supports the existence of PBR subtypes.