To understand more about the human inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) expression and the relationship between this gene and the family of other ITI heavy chain genes, an analysis of the structure of the ITIH3 gene and its promoter region was performed. This gene is a single copy gene, 14 kilobase pair in length and consists of 22 exons. ITIH3 shares highly conserved exon size and intron-exon borders with other ITI heavy chain genes. We determined that the human ITIH1, ITIH3, and ITIH4 genes are closely linked within a 45-kilobase pair. They are arranged in the order of H1-H3-H4, with the ITIH4 gene transcribed in the opposite direction. A model for the evolution of the ITI heavy chain gene family is presented that involves multiple rounds of gene duplication plus inversion events. The minimum promoter region (-135 to +75) is identified in HepG2 cells. The transient transfection study in various cell lines indicates that the activity of the ITIH3 promoter is not liver-specific. DNase I footprinting, mobility shift assays, and cotransfection experiments reveal a functional CCAAT/enhancer-binding protein site (C/EBP, -1344 to -1305) which interacts with C/EBPalpha and C/EBPbeta factors. The latter factors control the transcription of the ITIH3 gene positively.