Exposure to chlorpyrifos at doses that do not inhibit acetylcholinesterase can be followed by chronic illness in adults and developmental deficits in children. A mechanism to explain these effects is not available. Using mass spectrometry, we have found that chlorpyrifos oxon is a cross-linking agent. Pure proteins incubated with 1.5 mM chlorpyrifos oxon were diethylphosphorylated on lysine and tyrosine. The diethylphospho-lysine reacted with the carboxyl side-chain of aspartic and glutamic acid to form an isopeptide cross-link, releasing diethylphosphate in the process. Of the 14 proteins tested, 9 had cross-links between distinct proteins or between monomers of the same protein, whereas 8 had a cyclic structure created by joining side-chains of nearby residues through an isopeptide bond. The precursor lysine in the isopeptide bond was diethylphosphorylated on the ε-amino group. Tubulin was more susceptible to chlorpyrifos-oxon-induced cross-linking than the other proteins (10 cross-links in tubulin, 2 in human albumin). The role of diethylphospho-tyrosine was not examined. We hypothesize that the protein misfolding and protein cross-linking induced by exposure to chlorpyrifos oxon, via metabolism of chlorpyrifos, could disrupt function, particularly of tubulin, thus leading to chronic illness. Our proposed mechanism is hypothetical until the many questions it raises have been addressed.