5-Oxoprolinuria (pyroglutamic aciduria) resulting from glutathione synthetase (GSS) deficiency is an inherited autosomal recessive disorder characterized, in its severe form, by massive urinary excretion of 5-oxoproline, metabolic acidosis, haemolytic anaemia and central nervous system damage. The metabolic defect results in low GSH levels presumably with feedback over-stimulation of gamma-glutamylcysteine synthesis and its subsequent conversion to 5-oxoproline. In this study, we cloned and characterized the human GSS gene and examined three families with four cases of well-documented 5-oxoprolinuria. We identified seven mutations at the GSS locus on six alleles: one splice site mutation, two deletions and four missense mutations. Bacterial expression and yeast complementation assays of the cDNAs encoded by these alleles demonstrated their functional defects. We also characterized a fifth case, an homozygous missense mutation in the gene in an individual affected by a milder-form of the GSS deficiency, which is apparently restricted to erythrocytes and only associated with haemolytic anaemia. Our data provide the first molecular genetic analysis of 5-oxoprolinuria and demonstrate that GSS deficiency with oxoprolinuria and GSS deficiency without 5-oxoprolinuria are caused by mutations in the same gene.