A female infant who died 2.5 d after birth with hypoglycemia, lactic acidosis, and sudden multisystem failure was studied. Biochemical studies showed complex III and IV deficiency in liver, kidney, and muscle, with muscle most severely affected. Southern blot analysis of the patient's mitochondrial DNA did not reveal any deletions. Denaturing gradient gel analysis, which detects single base changes by differences in melting behavior, showed an extra band that was not seen in mitochondrial DNA from the mother, the mother's identical twin sister, or an unrelated normal subject. This extra band indicated heteroplasmy for a restriction fragment containing the apocytochrome b and transfer RNA(thr) genes. Sequencing revealed an A to G mutation at nucleotide 15923, the last base of the anticodon loop of the transfer RNA(thr) gene. The mutation lengthens the anticodon stem by added pairing and reduces the anticodon loop size from 7 to 5 nucleotides, potentially compromising transfer RNA(thr) function in translation and/or in processing the polycistronic RNA transcript. The patient's mother previously had a male infant who also died at 1.5 d postnatal, and both the mother and her twin have had multiple miscarriages. Amniocentesis for a genetic screen was performed on the mother's twin sister during a recent pregnancy; some of the cultured cells were made available for this study. The mutation was not found in the amniocytes or in umbilical cord blood obtained at birth; the baby was normal at birth and remains healthy. It is concluded that the mutation at nucleotide 15923 was most likely the cause of the fatal disease in the index case.(ABSTRACT TRUNCATED AT 250 WORDS)