We investigated the effect of pH and the additive monoethanolamine on (18)O-atom incorporation into the C-terminal carboxy group of peptide fragments during tryptic digestion in (18)O-labeled water. Although amidase activity was sufficient for digestion at pH 6-11, the second (18)O-atom incorporation at the carboxy oxygen site was inhibited at pH 11 or above. The addition of at least 50 mM monoethanolamine into the reaction mixture also inhibited the carboxy oxygen exchange without reduction in amidase activity. Therefore, tryptic digestion for (18)O-single labeling should be performed in 50 mM phosphate buffer (pH 11) containing 50 mM monoethanolamine. The production ratios of (18)O-single labeled peptides were over 85%, and these results were independent of amino acid sequence. We also investigated the linearity of the (18)O-single labeled to unlabeled ratio ((18)O(1)/(18)O(0)). The use of y ions for calculation of the (18)O(1)/(18)O(0) ratio gave a better correlation between the observed and theoretical (18)O(1)/(18)O(0) ratios in the range of 0.1 to 10 than did the use of precursor ions. In the analysis of a pseudobiomarker spiked into human serum, the present (18)O-single labeling method was found to be robust because it was not affected by incomplete LC separation. The present (18)O-single labeling method represents a useful tool for quantitative proteomics using nanoLC-ESI-MS/MS.