We have evaluated the immunoreactivity of 20 monoclonal antibodies (mAbs) directed against human apolipoprotein (apo)A-I with a panel of high density lipoproteins (HDL) from 13 mammalian species. The pattern of cross-reactivity showed that 20 mAbs had different specificity. While not all mAbs recognized apoA-I from all of the different species, the antigenicity of some sequences was well conserved. Thus, mAb A05 cross-reacted with all species except guinea pig and rat. In contrast, the mAb 4H1, which recognized residues 2-8, required a specific proline in position 3, as no immunoreactivity was found in the species missing this amino acid. Furthermore, the presence of a threonine residue in place of serine (in position 6) in the cynomolgus monkey was associated with a 20-fold loss of immunoreactivity in radioimmunometric assay with 4H1. As most of the epitopes were found in CNBr fragments 2 and 3, we sequenced these regions in four species (horse, goat, sheep, and cat) and analyzed the alignment of most known sequences to evaluate their consensus. Except for the rat and the chicken, considerable identity was observed. This permitted us to deduce the involvement of the residues in some antigenic epitopes. In the middle of apoA-I, a conservative mutation Asp103-->Glu was found sufficient to eliminate all reactivity of this epitope for A11 (residues 99-105 ... 12l6-132) in five species (rabbit, cow, goat, sheep, and rat). The residues essential to the expression of two other epitopes overlapping with A11 were also characterized. Edmundson-wheel representation of 18-residue repeated sequences of the different apoA-I species (for the eight amphipatic helices of residues 46-63, 68-85, 101-118, 123-140, 143-160, 167-184, 189-206, and 222-239) showed that secondary structure of apoA-I was more conserved than the antigenic epitopes. The N-terminal region, residues 1 to about 98, is rich in both strictly preserved sequences and epitope expression in most of the species surveyed. This evolutionary conservation of the N-terminal domain suggests an important yet unknown function.