The Golgi complex plays an important role in cholesterol trafficking in cells, and amyloid β-peptides (Aβs) alter cholesterol trafficking. The hypothesis was tested that fresh and aged Aβ-(1-42) would differentially modify Golgi cholesterol content in DINTC1 astrocytes and that the effects of Aβ-(1-42) would be associated with the region of the Golgi complex. Two different methods were used to determine the effects of Aβ-(1-42) on Golgi complex cholesterol. Confocal microscopy showed that fresh Aβ-(1-42) significantly increased cholesterol and that aged Aβ-(1-42) significantly reduced cholesterol content in the Golgi complex. Isolation of the Golgi complex into two fractions using density gradient centrifugation showed effects of aged Aβ-(1-42) similar to those observed with confocal microscopy but revealed the novel finding that fresh Aβ-(1-42) had opposite effects on the two Golgi fractions suggesting a specificity of Aβ-(1-42) perturbation of the Golgi complex. Phosphatidylcholine-phospholipase D activity, cell membrane cholesterol, and apolipoprotein E levels were associated with effects of fresh Aβ-(1-42) on cholesterol distribution but not with effects of aged Aβ-(1-42), arguing against a common mechanism. Extracellular Aβ-(1-42) targets the Golgi complex and disrupts cell cholesterol homeostasis, and this action of Aβ-(1-42) could alter cell functions requiring optimal levels of cholesterol.