Cytochrome P450IIB1,2 (nomenclature according to Nelson et al., DNA Cell Biol 12:1–51, 1993 and Volk et al., Neuroscience 42:215–235, 1991) immunoreactivity (P450‐IR) is associated with astrocytes both in vivo and in vitro. Although they are unevenly distributed throughout the brain with a preference for phylogenetically elder parts, no significant differences between astrocytes prepared from different brain regions were observed in astrocyte cultures. The percentage of strongly immunoreactive astrocytes decreased from 40% after 7 days in culture to 15% after 21 days. Essentially all astrocytes have a low but significant P450‐IR within this interval. Preembedding immunoelectron microscopy revealed peroxidase reaction products on the endoplasmic reticulum and on the outer membranes of mitochondrial and nuclear envelopes. Phenytoin (1 μM) added to the medium for 7 days significantly (1.22‐fold) increased the amount of total P450 in astrocyte homogenates as measured by spectrophotometry. Considerably more immunoreactive cells (1.5‐fold) were found in treated cultures than in controls. © 1994 Wiley‐Liss, Inc.