Chronic granulomatous disease (CGD) is a rare congenital disorder in which the patients' phagocytes fail to kill ingested microbes due to an inability to generate superoxide and other microbicidal oxygen derivatives. This inability is caused by mutations in one of the four components of the phagocyte-specific NADPH oxidase. A small subgroup of CGD patients has mutations in the CYBA gene that encodes the p22-phox subunit of the NADPH oxidase. This subunit forms, together with gp91-phox, a flavocytochrome b(558) heterodimer in the phagocyte plasma membrane. Expression of both subunits is required for normal expression of this heterodimer. Here, we report an autosomal recessive CGD patient with neutrophils that did not express flavocytochrome b(558) and did not generate superoxide upon activation. Analysis of genomic DNA revealed a 4-bp deletion at the exon-1/intron-1 boundary in CYBA (IVS1+4_7delAGTG). In the patient's cDNA, we found a low expression of an abnormal product, containing exon 1 extended by 79 nucleotides from intron 1, joined to exon 2. This extension is apparently caused by the activation of a cryptic donor splice site with a GT sequence at position 84-85 in intron 1. Both parents of the patient had the same mutation in their genomic DNA, in heterozygous form, but their cDNA contained exclusively the wild-type p22-phox cDNA sequence, indicating that the mutant mRNA was labile. This is, as far as we know, the first description of the molecular and clinical consequences of a donor splice site mutation in intron 1 of any gene reported so far.