Beta-2-microglobulin (beta2mu) production has been studied in 33 primary man-mouse hybrid clones and in 26 secondary man-mouse hybrid clones. These clones have also been examined for the presence of 15 human enzyme phenotypes. Karyotypic analyses have been carried out on clones. From these studies the following conclusions can be drawn: (1) Gene(s) determining human beta2mu production in humans are apparently syntenic with the MPI gene on chromosome 15. (2) Long-term fibroblast lines may be of limited use in mapping studies as chromosomal rearrangements frequently occur in these lines. (3) The gene(s) determining beta2mu production in humans segregate independently of chromosome 6. If the assignment of the genes determining HL-A alloantigens to chromosome 6 is correct, our results imply that beta2mu and HL-A alloantigens are determined by genes carried on different chromosomes, despite the fact that beta2mu forms an integral part of the HL-A molecule.