Under a tightly regulated expression mechanism, matrix metalloproteinases degrade extracellular matrix proteins and are though to play a role in injury repair and tumor metastasis in peripheral tissues. Little is known about the function of matrix metalloproteinases or agents that regulate their production in adult brain; however, it has been shown that the activity of a calcium‐dependent metalloproteinase is elevated in Alzheimer's hippocampus. The goals of this study were to determine whether cultured rat astrocytes produce matrix metalloproteinases and to identify agents that regulate protease activity. Enriched astrocyte cultures were prepared from brains of 1‐day‐old rat pups, and experiments were performed 13 days later. Gelatinase activity in astrocyte conditioned medium was determined using zymography with gelatin copolymerized with acrylamide in the gel. Under basal conditions after a 24‐h incubation, rat astrocytes produce gelatinases of 58 and 66 kDa. On stimulation of astrocytes with lipopolysaccharide, interleukin‐1α or ‐β, or tumor necrosis factor‐α for 24 h, a dose‐dependent increase in the activity of the 58‐ and 66‐kDa gelatinases and the induction of a 94‐kDa gelatinase occurred. All three astrocyte‐derived proteases showed maximal activity in the presence of millimolar levels of Ca²⁺, their activity was inhibited in the presence of 1,10‐phenanthroline, and their proenzymes were cleaved and activated after incubation with p‐aminophenylmercuric acetate. Using immunoblotting, immunopositive bands at the respective molecular sizes indicated that the 58‐kDa gelatinase was gelatinase A (matrix metalloproteinase 2) and the 94‐kDa activity was gelatinase B (matrix metalloproteinase 9). Induction of the 94‐kDa gelatinase by lipopolysaccharide was not influenced when interleukin‐1 receptor antagonist was included during the 24‐h incubation period; however, the antagonist completely blocked interleukin‐1β‐induced 94‐kDa activity and diminished the activity of the 58‐ and 66‐kDa gelatinases. Dexamethasone inhibited both lipopolysaccharide and interleukin‐1β stimulation of the 94‐kDa gelatinase. These results indicate that cytokines regulate matrix metalloproteinase expression in cultured rat astrocytes. Because astrocytes become “activated” (are hypertrophic and express increased levels of glial fibrillary acidic protein) in the presence of several inflammatory cytokines, it is possible that these astrocyte‐derived enzymes contribute to the activation process and may participate in tissue remodeling after brain injury.