Coproporphyrinogen oxidase is a mitochondrial heme-biosynthetic enzyme that converts coproporphyrinogen to protoporphyrinogen. Inherited deficiency of this enzyme causes the human genetic disease hereditary coproporphyria. Recently, we isolated, sequenced and expressed the cDNA encoding human coproporphyrinogen oxidase. This allowed us to investigate the nature of the defect leading to a profound deficiency of coproporphyrinogen oxidase in a patient with homozygous hereditary coproporphyria. Using reverse-transcription, amplification of the cDNA and direct sequencing of the amplified products, we found a point mutation resulted in an arginine to tryptophane substitution (R231W). Expression studies of normal and mutated cDNAs in a bacterial system demonstrated that this substitution resulted in the synthesis of an unstable protein with a residual catalytic activity. This is the first mutation to be found at the coproporphyrinogen oxidase locus. Furthermore, three common polymorphisms within the coproporphyrinogen oxidase gene were detected. Two DNA polymorphisms resulted in amino acids changes (H172N and V194I) and the third one was silent (E230E).