Insects respond to microbial infection by the rapid and transient expression of several genes encoding antibacterial peptides. In this paper we describe a powerful technique, two-dimensional difference gel electrophoresis, that, when combined with mass spectrometry, can be used to study the immune response of Drosophila melanogaster at the protein level. By comparatively analyzing the hemolymph proteome of 2,000 third-instar Drosophila larvae, we identified 10 differential proteins that appear in the fruit fly hemolymph very early after an immune-challenge with lipopolysaccharides. These proteins can be assigned to the immune response, because they are not induced after sterile injury. Reduction of intergel variability or quantification problems related to conventional two-dimensional electrophoresis and improvement of image analysis were achieved by the use of two fluorescent dyes to label the two different protein samples. Some of the immune-induced proteins, such as thioester-containing protein 2, can be assigned to specific aspects of the immune response; others were already reported as being involved in stress response. An immune-induced protein (CG18594) is homologous to a mammalian serine protease inhibitor that mediates the mitogen-activated protein kinase and the NF-κB signaling pathways. In addition, a number of proteins that had not been associated with the immune response before were isolated and identified, and some of these were still present in the hemolymph 4 h after injury. Determining the function of all of these immune-induced proteins represents an exciting challenge for increasing our knowledge of insect immunity.