The physiological role of chromium (III) in diabetes mellitus has been an area of inconclusive research for many years. It is of great interest to explore the interactions made by chromium (III) to get a better insight into their role in glucose metabolism. To understand the molecular basis of chromium action we have carried out spectroscopic and crystallographic investigations on the binding of Cr(III)Salen with insulin, as Cr(III)-Salen is reported to result in the enhancement of insulin activity. The Cr(III)-insulin complex formation has been characterised at two pHs, viz., 3.5 and 9.0 using UV-Vis and fluorescence studies. The crystallographic analysis of Cr(III)-Salen soaked cubic insulin crystals, using anomalous difference Fourier method, revealed B21 Gin to be the binding site for chromium (III). (c) 2008 Elsevier Inc. All rights reserved.