Monocytes recruited into tissues from peripheral blood differentiate into macrophages, which are critical in the pathogenesis of many diseases. There is limited data concerning the global changes in the expression of genes during monocyte to macrophage differentiation, and how the patterns of change identify the mechanism contributing to macrophage differentiation or function. Employing microarray technology, we examined the transcriptional profile of in vitro adherence-induced differentiation of primary human monocytes into macrophages. We found the significant up regulation of genes contributing to the functions of macrophages, including those regulating to immunity and defense; lipid, fatty acid and steroid metabolism; cell adhesion, carbohydrate metabolism; amino acid metabolism and endocytosis. In contrast, the vast majority of transcription factors affected were down regulated during monocyte to macrophage differentiation, suggesting that transcriptional repression may be important for the transition from monocytes to macrophages. However, a limited number of transcription factors were up regulated, among these was C/EBPalpha, which may contribute to differentiation by regulating down stream genes, which are a characteristic of differentiated macrophages. These observations suggest that examination of the transcriptional profile in monocytes and macrophages in patients may identify relevant therapeutic targets in diseases mediated by macrophages.