Exposure to organophosphorus (OP) agents can lead to learning and memory deficits. Disruption of axonal transport has been proposed as a possible explanation. Microtubules are an essential component of axonal transport. In vitro studies have demonstrated that OP agents react with tubulin and disrupt the structure of microtubules. Our goal was to determine whether in vivo exposure affects microtubule structure. One group of mice was treated daily for 14 days with a dose of chlorpyrifos that did not significantly inhibit acetylcholinesterase. Beta-tubulin from the brains of these mice was diethoxyphosphorylated on tyrosine 281 in peptide GSQQY(281)RALTVPELTQQMFDSK. A second group of mice was treated with a single sublethal dose of chlorpyrifos oxon (CPO). Microtubules and cosedimenting proteins from the brains of these mice were visualized by atomic force microscopy nanoimaging and by Coomassie blue staining of polyacrylamide gel electrophoresis bands. Proteins in gel slices were identified by mass spectrometry. Nanoimaging showed that microtubules from control mice were decorated with many proteins, whereas microtubules from CPO-treated mice had fewer associated proteins, a result confirmed by mass spectrometry of proteins extracted from gel slices. The dimensions of microtubules from CPO-treated mice (height 8.7 +/- 3.1 nm and width 36.5 +/- 15.5 nm) were about 60% of those from control mice (height 13.6 +/- 3.6 nm and width 64.8 +/- 15.9 nm). A third group of mice was treated with six sublethal doses of CPO over 50.15 h. Mass spectrometry identified diethoxyphosphorylated serine 338 in peptide NS(338)NFVEWIPNNVK of beta-tubulin. In conclusion, microtubules from mice exposed to chlorpyrifos or to CPO have covalently modified amino acids and abnormal structure, suggesting disruption of microtubule function. Covalent binding of CPO to tubulin and to tubulin-associated proteins is a potential mechanism of neurotoxicity.