Chromium has been observed to play a role in maintaining proper carbohydrate and lipid metabolism of mammals. One of the potentially biological active forms of chromium in vitro is low-molecular-weight chromium-binding substance (LMWCr), which has been proposed to amplify the insulin cascade by binding with insulin receptor. LWMCr is a small bio-molecule (<1500 Da) containing a carboxylate-rich polypeptide with four bound chromic ions. LMWCr's (according to its amino acid composition and mass spectrum) were successfully isolated from chicken and alligator livers, as well as from human urine, using a modified method. The extreme hydrophilicity of the peptide and the tightly bound Cr(III) are two major hurdles to produce a stable end-product for mass spectrometry (MS) or high-performance liquid chromatography (HPLC) analysis. Treating bovine LMWCr with trifluoroacetic acid and application to a graphite powder micro-column was used to generate a heptapeptide fragment, and the peptide sequence was analyzed by mass spectrometry (MS) and tandem MS (MS/MS). Two candidate sequences, EEEEGDD and EEEGEDD, were identified; the mass spectrum of the former sequence is more similar to that of the LMWCr fragment. Langmuir isotherm and Hill plots were used to analyze the binding constants of chromic ions to synthetic peptides similar in composition to LMWCr and apoLMWCr. The sequence pEEEEGDD can bind 4 chromic ions per peptide as apoLMWCr does, while the other sequences examined only bind two chromic ions. Studies to further elucidate the structure of LMWCr are ongoing.