Hereditary haemochromatosis is a common inherited disorder leading to excessive accumulation of iron in various organs. Two missense substitutions at the HFE-gene have recently been associated with the disease, 187C G and 845G → A (mutations H63D and C282Y, respectively). We present a simple, rapid PCR–SSCP multiplex screening method allowing the simultaneous detection of both substitutions. Furthermore, testing the method on 420 Danish blood donors revealed the presence of a hitherto undetected third substitution in 13 individuals. The new substitution, a 193A → T transversion, affects codon 65 changing the code for serine to that of cysteine (S65C). It may thus have functional consequences for the HLA class protein encoded by the HFE-gene. The allele frequencies observed were: H63D 14.8%, C282Y 6.2% and S65C 1.5%, which for the two former alleles are in agreement with frequencies reported for other North European population samples.