The serotype H immobilization antigen (H i-ag) gene of Paramecium tetraurelia was isolated from a genomic library by screening with the H i-ag transcript. The gene is 7.5 kb long and contains no introns. Similar to other i-ag genes, it does not undergo rearrangement or duplication upon expression. In contrast, only the H i-ag gene appears to be non-adjacent to a telomere. Sequence comparisons between the H, A, and C i-ag genes of stock 51 and the G i-ag gene of stock 156 identify conserved upstream sites and extensive homology in the 3 non-coding region. Several of these sites may be involved in regulating the expression of the i-ags. Segments of amino acid homology among these genes are also predicted by the DNA sequences.