The family of roundabout (Robo) proteins is related to the transmembrane receptors and plays a major role in the process of axonal guidance in neurogenesis. It has recently been shown that Robo proteins are also associated with tumor angiogenesis with Slit2 acting as the corresponding ligand. The aim of this study was to validate the differential expression by means of microarray analysis and real-time PCR and to analyze the in situ expression of Robo1 and Robo4 in colorectal cancer. Quantitative analyses of Robo1, Robo4 and Slit2 mRNA expression measured by large scale gene expression studies (Affymetrix U133A) showed a significant up-regulation of Robo1 in tumor vs. normal tissue, whereas Robo4 and Slit2 showed no significant deregulation. For subsequent real-time PCR experiments, paired colorectal tissue samples from cancerous and corresponding non-cancerous tissues were obtained from 50 colorectal cancer patients who underwent surgical resection. Robo1 mRNA overexpression in cancerous tissue compared with normal counterparts was observed in 80% of the patients with a 4-fold expression in 45% and a 12-fold expression in 15%. For Robo4, an up-regulation was detected in >70% (36/50). For Slit2, no differential expression was observed. The overexpression of Robo1 and Robo4 in tumor vs. normal tissue was verified using real-time PCR. The histological analysis revealed an expression of Robo1 mainly in tumor cells, whereas Robo4 is located primarily in endothelial cells of tumor vessels. Therefore, the Robo proteins provide potential target structures for the anti-tumorigenic and anti-angiogenic therapy of colorectal carcinoma.