Arginine:glycine amidinotransferase (AGAT) is the enzyme responsible for the conversion of arginine and glycine into guanidinoacetate (GuAc) and ornithine in creatine biosynthesis. AGAT deficiency was recently described in two patients by Item et al. (1). These two patients [for whom the clinical details were first described by Bianchi et al. (2)] are mentally retarded and have severe creatine deficiency in the brain and decreased urinary GuAc. Several methods for measuring AGAT activity have been published, but these methods are nonspecific because the measured ornithine formed during the assay can be of different origin (3)(4) or they are impracticable because they use radioactivity (1). We developed a new method in lymphocytes and lymphoblasts that uses stable-isotope-labeled substrates. We used l-[guanido-15N2]arginine and [U-13C,15N]glycine as substrates and analyzed the enzyme product [1,2-13C2,15N3]GuAc (after derivatization) by gas chromatography–mass spectrometry using [1,2-13C2]GuAc as internal standard. We applied this method to measure enzyme activities in lymphocytes and lymphoblasts of controls and in lymphoblasts from a patient affected with AGAT deficiency. For lymphocyte isolation, whole venous blood from 16 control individuals was drawn into acid-citrate dextrose and stored at room temperature up to 48 h. Written consent was obtained from each participant. Lymphoblast lines derived from six control individuals were maintained in RPMI-1640 supplemented with 100 mL/L fetal bovine serum and 10 mL/L penicillin/streptomycin. The lymphoblast lines used in this study as controls were originally obtained for carrier screening. No defect was found, and the samples were anonymized. Unless otherwise stated, all chemicals and reagents were purchased from Sigma, Baker, Merck, or Pierce. [U- …