A new mitochondrial disease associated with mitochondrial DNA (mtDNA) heteroplasmy was identified in a family. Four family members exhibited a variable combination of symptoms including developmental delay, retinitis pigmentosa, dementia, seizures, ataxia, proximal neurogenic muscle weakness, and sensory neuropathy, all maternally inherited. No histochemical evidence of mitochondrial myopathy was found. Blood and muscle samples from patients contained two mtDNA populations, one of which had a previously unreported AvaI restriction site. Sequence analysis revealed a point mutation at nucleotide 8993, causing an amino acid change from leucine to arginine in subunit 6 of the mitochondrial H+ -ATPase. Clinical severity correlated with the amount of mutant mtDNA, which was present in small quantities in healthy relatives. The mutation at position 8993 is highly conserved in other species and results in a hydrophilic amino acid replacing a hydrophobic one, potentially affecting enzyme structure and function. The presence of two mtDNA populations (heteroplasmy) in this maternal lineage is notable, as heteroplasmy has not been previously observed in control subjects. This study highlights the importance of mtDNA heteroplasmy in disease and suggests a link between mtDNA mutations and neurologic symptoms. The findings contribute to understanding the genetic basis of mitochondrial diseases and the role of mtDNA in human health.A new mitochondrial disease associated with mitochondrial DNA (mtDNA) heteroplasmy was identified in a family. Four family members exhibited a variable combination of symptoms including developmental delay, retinitis pigmentosa, dementia, seizures, ataxia, proximal neurogenic muscle weakness, and sensory neuropathy, all maternally inherited. No histochemical evidence of mitochondrial myopathy was found. Blood and muscle samples from patients contained two mtDNA populations, one of which had a previously unreported AvaI restriction site. Sequence analysis revealed a point mutation at nucleotide 8993, causing an amino acid change from leucine to arginine in subunit 6 of the mitochondrial H+ -ATPase. Clinical severity correlated with the amount of mutant mtDNA, which was present in small quantities in healthy relatives. The mutation at position 8993 is highly conserved in other species and results in a hydrophilic amino acid replacing a hydrophobic one, potentially affecting enzyme structure and function. The presence of two mtDNA populations (heteroplasmy) in this maternal lineage is notable, as heteroplasmy has not been previously observed in control subjects. This study highlights the importance of mtDNA heteroplasmy in disease and suggests a link between mtDNA mutations and neurologic symptoms. The findings contribute to understanding the genetic basis of mitochondrial diseases and the role of mtDNA in human health.