Rationale: There is a need in imaging mass spectrometry to use the acquired isotope distribution to unequivocally determine the identity of a peptide ion. A way of achieving unambiguous differentiation of ions from protonated peptides from other [M+H] + ions in a tissue would be via the direct on-tissue incorporation of 18O into peptides. Methods: Tissues were first digested with trypsin for 3h at 37°C in a humidified chamber. For the 18O-labelling of digested peptides 1μL of H 2 18O/50mM ammonium acetate (at pH 6.75) was added to the array of tryptic spots and incubated at room temperature for 20min. α-Cyano-4- hydroxycinnamic acid was used as a matrix modifier. The mass spectral analysis of tissue sections was carried out using a matrix-assisted laser desorption/ionisation tandem time-of-flight (MALDI-TOF-TOF) instrument. Results: On-tissue incorporation of 18O into peptides cannot be carried out during the digestion step inside a humidified chamber. After tissue digestion for 3h at 37°C in an humidified chamber, 18O labelling was carried out for 20min at room temperature (no humidified chamber). No trypsin was needed to enhance the labelling. Conclusions: For first time the feasibility of 18O-labelling of peptides in situ for tissues has been demonstrated. The method decouples protein digestion from peptide labelling and is performed in sequential steps. Furthermore, we observed that 18O incorporation produces characteristic isotopic peptide distributions, thus making facile distinguishing peptides from other tissue molecular components that ionise in the MALDI ion source.