Hemochromatosis (HH) is an inborn error of iron metabolism, frequent among Caucasians, characterized by progressive iron loading that, if untreated, causes high morbidity and death. HLA-H, a putative HH gene, has recently been isolated. The large majority of patients so far studied are homozygous for a single mutation, which results in a cysteine-to-tyrosine substitution at amino acid 282 of the protein. A second, less frequent, variant, His63Asp, has an undefined role in the pathogenesis of the disease. Here we report that the Cys282Tyr change accounts for 69% of HH chromosomes in a series of 75 unrelated Italian patients who fulfilled well-defined criteria for HH diagnosis. Sixty-four percent of patients were Cys282Tyr homozygous, 10% were heterozygous, and 21% carried the normal allele. The same mutation was rare in normal controls. The His63Asp variant was less frequent but had a similar frequency among affected and normal chromosomes. Subjects without two copies of the Cys282Tyr change were both isolated patients and individuals from families with a 6p-linked disease. Mutation analysis of the HLA-H gene, carried out by RNA-SSCP in the latter patients, did not reveal any significant nucleotide abnormality in coding sequences and intron-exon boundaries. The absence of mutations in HLA-H gene was confirmed in three cases by direct sequencing. Major deletions or rearrangements of the gene were excluded by Southern blotting. The existence of patients with clinical and histological features of HH, but without mutations in HLA-H gene, suggests that in Italy the disease is more heterogeneous than reported in northern Europe.