A rare high oxygen affinity hemoglobin variant was identified in a 22-year-old male patient from Napoli (Naples, Italy) affected by erythrocytosis. A detailed structural characterization of the variant hemoglobin was carried out, both at the protein and DNA levels essentially by mass spectrometric procedures and allele-specific amplification techniques. The amino acid substitution was determined by liquid chromatography tandem mass spectrometric analysis of the tryptic digest as beta36(C2)Pro-->His; the corresponding DNA mutation was identified as C-->A at the second position of codon 36 of the beta chain (CCT-->CAT). These variations identified the presence of Hb Vila Real, described only once before in a Portuguese woman. Haplotype analysis of DNA polymorphisms showed that the beta-globin gene of Hb Vila Real was associated with haplotype I.