Extensive incorporation of oxygen-18 at position O4 of the pyrimidine nucleus results from exchange between H218O and nucleosides or bases in 1N HC1 at 100 degrees. The reaction is hindered by substitution at C-5 with the greatest effect shown in pseudouridine (R = ribosy1) and the least in uridine (R = H). Maximum incorporation in the latter compound was 94%, and in uricil was 98%. The method is experimentally simple and the incorporation is readily monitored by mass spectrometry.