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BACKGROUND: Approximately 3% of patients exposed to iodinated contrast media develop delayed hypersensitivity reactions. OBJECTIVE: We wanted to better understand the molecular basis of contrast media cross-reactivity. METHODS: Cross-reactivity was assessed by skin testing and measurement of T-cell activation (CD69 upregulation) and proliferation ((3)H-thymidine uptake, 5,6-carboxyfluorescein diacetate succinimidyl ester staining) of PBMCs, T-cell lines, and T-cell clones of 2 patients with delayed hypersensitivity reactions to iohexol and iomeprol, respectively. Thirteen different contrast media and potassium iodide were compared. RESULTS: Skin testing and analyses of PBMCs, T-cell lines, and clones showed broad cross-reactivity in both patients. Broad as well as more restricted cross-reactivity patterns were found in iohexol-specific and iomeprol-specific CD4(+) T-cell clones, whereas 1 iomeprol-specific CD8(+) T-cell clone showed no cross-reactivity at all. The reactivity to equimolar concentrations of iohexol and its dimer iodixanol was very similar, suggesting that the dimer was not more stimulatory than its monomer. Consistently low reactivity to iobitridol was found in both patients, but never to iodide. A frequency analysis of contrast medium-specific peripheral T cells gave values between 0.6 % (iomeprol) and 0.05 % (iobitridol). CONCLUSION: Clinically observed cross-reactivity between different contrast media is a result of the presence of contrast media-specific T cells, some of which show a broad cross-reactivity pattern. Iodide ions, known to be present at low concentration in contrast media solutions, do not seem to be the causative moiety. CLINICAL IMPLICATIONS: Detailed in vitro analysis might help identify noncross-reactive contrast media.