The serine protease inhibitor alpha 1-antichymotrypsin (ACT) has been shown to be tightly associated with the amyloid found in plaque cores and blood vessels in the brains of patients with Alzheimer's disease (AD). Although the ACT found in plaques could be derived from the high levels of ACT in serum, previous Northern analysis revealed that ACT mRNA is produced locally in AD gray matter at much higher levels than in control gray matter. To determine which brain cells express ACT mRNA, we conducted in situ hybridization with 35S-labeled cRNA probes on hippocampal sections from four AD and three control cases. To identify astrocytes unequivocally, some of the hybridized sections were immunostained for glial fibrillary acidic protein, which is astrocyte-specific. Our results showed numerous astrocytes that were intensely labeled by the probe for ACT mRNA throughout the subicular gray matter of the AD cases. In contrast, astrocytes in control gray matter were rarely labeled by the probe for ACT mRNA. Examination of plaque cores in the AD subiculum showed that some astrocytes intensely labeled by the probe for ACT mRNA were closely associated with virtually every plaque core. Our results also showed many astrocytes in both AD and control white matter that were intensely labeled by the probe for ACT mRNA, and a small fraction of the astrocytes in a juvenile cerebellar astrocytoma that we examined were found to produce high levels of ACT mRNA. In every area in which astrocytes expressing ACT mRNA were found, astrocytes producing no detectable ACT message were also present. Our findings indicate that astrocytes produce the increased ACT mRNA in AD gray matter observed by Northern analysis, but they also show that ACT mRNA expression by astrocytes is not unique to AD. The presence of astrocytes expressing ACT mRNA near, and extending processes towards, plaque cores strongly suggests that some if not all of the ACT associated with amyloid plaque cores is produced by astrocytes surrounding the cores.