Choline completely deuterated in the methyl groups has been incorporated into two mammalian cell systems. The first was the chemically transformed mouse fibroblast LM cell line, grown in suspension culture. The second system consisted of mitochondria from Sprague-Dawley rats which were fed upon a choline-deficient diet enriched with deuterated choline chloride. Two classes of deuterium nuclear magnetic resonance (NMR) signal were obtained from each system. The first class of signal exhibited no residual quadrupole coupling constant and was assigned to the natural abundance of 2H in water, together with free labeled choline. The second class of signal had a residual quadrupole coupling of about 1 HKz and was assigned to 2H-labeled choline headgroups of phospholipids. These results are in conflict with a previous study of deuterated rat liver mitochondrial membranes. Deuterium magnetic resonance spectra can be obtained on mammalian membranes that have incorporated specific deuterium-labeled compounds and, therefore, provides a very powerful method for studying the dynamic structure of membranes.