We studied the metabolism in the male F344 rats of N-nitrosomorpholine and of 3,3,5,5-tetradeutero-N-nitrosomorpholine ; the latter is less carcinogenic and less mutagenic than is N-nitrosomorpholine. alpha-Hydroxylation (3- or 5-hydroxylation) of N-nitrosomorpholine by liver microsomes and a reduced nicotinamide adenine dinucleotide phosphate-generating system produced (2-hydroxyethoxy)acetaldehyde, which was identified as its 2,4-dinitrophenylhydrazone derivative. When we administered N-nitrosomorpholine to rats i.p., we did not detect (2-hydroxyethoxy)acetaldehyde in the urine, but we did identify (2-hydroxyethoxy)acetic acid (16% of the dose). We also identified N-nitroso(2-hydroxyethyl)glycine (33% of the dose) from beta-hydroxylation (2- or 6-hydroxylation), N-nitrosodiethanolamine (12%), and unchanged N-nitrosomorpholine (1.5%) in the urine. The deuterated analogs of the above metabolites were isolated from the urine of rats treated with 3,3,5,5-tetradeutero-N-nitrosomorpholine in yields as follows: (2-hydroxyethoxy)acetic acid (3.4%); N-nitroso(2-hydroxyethyl)glycine (37%); N-nitrosodiethanolamine (12%); N-nitrosomorpholine (0.4%). These data demonstrates that deuterium substitution in the alpha-positions of N-nitrosomorpholine caused a decrease in the extent of alpha-hydroxylation and indicate that alpha-hydroxylation is the mechanism of activation of N-nitrosomorpholine.