Diabetic nephropathy is characterized by accumulation of mesangial matrix. Glucose-induced inhibition of matrix-degrading enzymes such as collagenases is believed to contribute to matrix accumulation. We have previously demonstrated that 72 kDa type IV collagenase activity is decreased in the rat mesangial cells cultured in high glucose media [ Diabetes 1995;44:929–935]. The present studies were designed to investigate if the cytokine transforming growth factor-β<sub>1</sub> (TGF-β<sub>1</sub>) mediates this effect of glucose. Type IV collagenases degrade type IV collagen as well as gelatin (denatured collagen) and are thus also called gelatinases. They belong to the family of matrix metalloproteinases (MMPs); MMP activity is controlled by tissue inhibitors of metalloproteinases (TIMPs). The activity of 72 kDa type IV collagenase, also known as matrix metalloproteinase-2 (MMP-2), was assessed using three methods: (1) fluoresceinated gelatin degradation assay to detect free enzyme activity (activity which is present in excess of TIMP-inhibited activity); (2) zymography to measure total (free + TIMP-bound) enzyme activity; (3) ELISA using specific antibodies to measure MMP-2 levels. TGF-β<sub>1</sub> and TIMP-2 levels were also determined by ELISA. Incubation of primary cultures of rat mesangial cells for 5 days in 30 vs. 5 m M glucose resulted in a 3-fold increase in production of total TGF-β<sub>1</sub>, a significant decrease in MMP-2 activity and immunoreactive MMP-2 levels, and an increase in TIMP-2 levels. Addition of exogenous TGF-β<sub>1</sub> to mesangial cells incubated in 5 m M glucose replicated the high glucose effect by producing a significant decrease in MMP-2 levels with a concurrent increase in TIMP-2 levels. Furthermore, glucose-induced inhibition of MMP-2 activity was completely blocked by neutralization of TGF-β<sub>1</sub> with anti-TGF-β<sub>1</sub> antibody. We conclude that the decrease in MMP-2 activity induced by glucose loading is mediated via TGF-β<sub>1</sub>.