A major impediment to CNS repair is the glial scar, which forms following damage and is composed mainly of ramified, ‘reactive’ astrocytes that inhibit neuronal regrowth. The transition of astrocytes into this reactive phenotype (reactive gliosis) is a potential therapeutic target, but glial scar formation has proved difficult to study in monolayer cultures because they induce constitutive astrocyte activation. Here we demonstrate a 3D collagen gel system in which primary rat astrocytes were maintained in a persistently less reactive state than comparable cells in monolayer, resembling their status in the undamaged CNS. Reactivity, proliferation and viability were monitored and quantified using confocal, fluorescence and time-lapse microscopy, 3D image analysis, RT–PCR and ELISA. To assess the potential of this system as a model of reactive gliosis, astrocytes in 3D were activated with TGFβ1 to a ramified, reactive phenotype (elevated GFAP, Aquaporin 4, CSPG, Vimentin and IL-6 secretion). This provides a versatile system in which astrocytes can be maintained in a resting state, then be triggered to undergo reactive gliosis, enabling real-time monitoring and quantitative analysis throughout and providing a powerful new tool for research into CNS damage and repair. Copyright © 2009 John Wiley & Sons, Ltd.