A gas chromatographic-mass spectrometric method has been developed for the determination of N-7-[ 2H 3]methyl guanine in urine in the presence of large natural levels of N-7-methyl guanine. Urine is fractionated on heptanesulfonic acid-treated C-18 Sep-pak cartridges, followed by derivatization to give a volatile N-heptafluorobutyryl- O 6-2,3,4,5,6-pentafluorobenzyl derivative which is separated on an SE52 fused silica capillary column. Using N-7-ethyl guanine as an internal standard, the total amount of N-7-methyl guanine is determined by gas chromatography-flame ionization detection. The percentage of N-7-[ 2H 3]methyl guanine is then measured by gas chromatography-mass spectrometry, enabling the amount of deuterated base to be determined. Preliminary experiments with [ 2H 3]methyl methanesulfonate in rats showed measurable excretion of N-7-[ 2H 3]methyl guanine. 4-(Di[ 2H 3]methylamino)antipyrine alone gave no detectable amount of alkylated base, but coadministration of nitrite resulted in excretion of deuterated N-7-methyl guanine.