Inter-alpha-inhibitor (I alpha I) and related molecules in human are comprised of three evolutionarily related, heavy (H) chains and one light (L) chain, also termed bikunin. The latter originates from a precursor molecule that is cleaved to yield the bikunin and another protein designated alpha-1-microglobulin (A1m). The four H and L chains are encoded by four distinct genes designated H1, H2, H3, and L. The L and H2 genes are localized onto human chromosomes (chr) 9 and 10, respectively, whereas the H1 and H3 genes are tandemly arranged on chr 3. Mouse poly(A)+ RNAs or endonuclease-restricted mouse DNA were analyzed by standard and pulsed-field gel electrophoresis (PFGE) techniques in agarose gels and blot-hybridized with human H1, H2, H3 or L cDNA probes. The variable sized transcripts and unique restriction fragment patterns detected with each probe indicate that four genes, including one common L gene for A1m and bikunin also exist in mouse. The co-migration of H1- and H3-hybridizing fragments on PFGE suggests that the mouse H1 and H3 genes are also tandemly arranged. An Msp I restriction fragment length polymorphism (RFLP) in the mouse L gene (proposed symbol, Intin-4) links this gene to other genes already mapped at mouse Chr 4 near the brown (b) locus, a homologous region to the human chr 9q32-34 band where the human I alpha I L gene is located. Therefore, a similar number and arrangement of I alpha I genes is found in mouse and human, including the triplication of an H gene ancestor. These results point to an ancient origin of this complex set of genes.