Organochlorine compounds have been linked to increased risk of several cancers. Despite reductions in their use and fugitive release, they remain one of the most important groups of persistent pollutants to which humans are exposed, primarily through dietary intake. We designed a case–control study to assess the risk of colorectal cancer with exposure to these chemicals, and their potential interactions with genetic alterations in the tumors. A subsample of cases (n = 132) and hospital controls (n = 76) was selected from a larger case–control study in Barcelona, Catalonia, Spain. We measured concentrations in serum of several organochlorines by gas chromatography. We assessed point mutations in K-ras and p53 genes in tissue samples by polymerase chain reaction/single-strand conformation polymorphism and assessed expression of p53 protein by immunohistochemical methods. An elevated risk of colorectal cancer was associated with higher serum concentrations of mono-ortho polychlorinated biphenyl (PCB) congeners 28 and 118. The odds ratio for these mono-ortho PCBs for middle and higher tertile were, respectively, 1.82 [95% confidence interval (CI), 0.90–3.70] and 2.94 (95% CI, 1.39–6.20). α-Hexachlorocyclohexane, hexachlorobenzene, and p,p′-DDE (4,4′-dichlorodiphenyltrichloroethene) showed nonsignificant increases in risk. Risk associated with mono-ortho PCBs was slightly higher for tumors with mutations in the p53 gene but was not modified by mutations in K-ras. Mono-ortho PCBs were further associated with transversion-type mutations in both genes. These results generate the hypothesis that exposure to mono-ortho PCBs contributes to human colorectal cancer development. The trend and magnitude of the association, as well as the observation of a molecular fingerprint in tumors, raise the possibility that this finding may be causal.