Innate immunity is based on the recognition of cell surface molecules of infecting agents. Microbial substances, such as peptidoglycan, lipopolysaccharide (LPS) and -1,3-glucans, produce functional responses in Drosophila hemocytes that contribute to innate immunity. We have used two-dimensional gel electrophoresis and mass spectrometry to resolve LPS-induced changes in the protein profile of a Drosophila hemocytic cell line. We identified 24 intracellular proteins that were up-or down-regulated, or modified, in response to immune challenge. Several proteins with predicted immune functions, including lysosomal proteases, actin-binding/remodeling proteins as well as proteins involved in cellular responses to oxidative stress, were affected by the immune assault. Intriguingly, a number of the proteins identified in this study have recently been implicated in phagocytosis in higher vertebrates. We suggest that phagocytosis is activated in Drosophila hemocytes by the presence of microbial substances, and that this activation constitutes an evolutionarily conserved arm of innate immunity. In addition, a number of proteins involved in calcium-regulated signaling, mRNA processing and nuclear transport were affected, consistent with a possible role in reprogramming of gene expression. In conclusion, the present proteome analysis identified many proteins previously not linked to innate immunity, demonstrating that differential protein profiling of Drosophila hemocytes is a valuable tool for identification of new players in immune-related cellular processes.