Bioorthogonal reactions are valuable tools for the selective labeling and imaging of natural products and proteins. Here, we present the reaction between isonitriles and chlorooximes as a ligation that proceeds quickly (k approximate to 1 M-1 s(-1)) and with high chemoselectivity in an aqueous environment. Imaging of metabolically labeled cell surface glycans underlined the tolerance of the ligation to common functional groups in cellular systems. Live-cell dual-labeling experiments revealed that the isonitrile-chlorooxime ligation is orthogonal to the strain-promoted azide-alkyne cycloaddition.