Adipose tissue plays a crucial endocrine role in controlling whole body glucose homeostasis and insulin sensitivity. Given the substantial rise in obesity and obesity-related diseases such as diabetes, it is important to understand the molecular basis of adipocyte differentiation and its control. Many studies have successfully exploited gene array technology to monitor changes in the profile of expressed genes during adipocyte differentiation, although this method only measures changes at the level of individual mRNA species. Using two-dimensional polyacrylamide gel electrophoresis, high-throughput image analysis, and candidate picking coupled with sequencing mass spectrometry, we have followed the changes in protein expression profile that occur during the differentiation of 3T3-L1 fibroblasts into adipocytes in response to dexamethasone, isobutyl methyl xanthine and insulin, or to the PPARgamma agonist, ciglitazone. Using this technique we have found alterations in the profile of over 2000 protein species during adipogenesis. Our studies reveal previously unknown alterations during adipogenesis in the expression or mobility (on sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of coactosin, which promotes actin filament destabilization, several signalling molecules, including RhoGDI-1, RhoGDI-2 and EHD1, and NEDD5 a protein involved in cytokinesis.