The sequence database of HLA class I genes focuses on the coding sequences, the exons. Limited information is available on the non-coding sequences of the different class I alleles. In this study we have determined the intron 4 nucleotide sequence of at least one representative of each major allelic group of HLA-A, -B and -C. The intron 4 sequences were determined for 27 HLA-A, 81 HLA-B and 30 HLA-C alleles by allele-specific sequencing, using primers located in adjacent exons and introns. The sequences revealed that the length of intron 4 varies with a minimum of 93 and a maximum of 124 nucleotides as a result of insertions and deletions. There were remarkable similarities and differences within HLA-A, -B and -C, as well as between them. Within HLA-A, a deletion of three nucleotides was detected in several HLA-A alleles. The HLA-B alleles could be divided into two groups with one group having a deletion of 11 nucleotides compared with the second group. Within HLA-C, all Cw*07 alleles showed remarkable differences with the other Cw alleles. Cw*07 had an insertion of three nucleotides, shared only by the Cw*17 group. Moreover, Cw*07 was found to have an aberrant nucleotide sequence. Differences between HLA-A, -B and -C alleles were also observed. Remarkable was the deletion of 20 nucleotides in all HLA-A and -B alleles compared with HLA-C, whereas the HLA-A alleles showed an insertion of one nucleotide and a deletion of three nucleotides compared with HLA-B and -C. Furthermore, 32 different polymorphic positions were detected between HLA-A, -B and -C.