Human alpha 1-antitrypsin ( alpha-1-AT;Pi) production was analyzed in 11 primary mouse hepatoma-human lymphoid cell hybrids and in 14 secondary rat hepatoma-human fetal liver fibroblast hybrids. The presence of human alpha-1-AT was determined by Laurell immunoelectrophoresis of concentrated and isotopically labeled supernatant medium. Human alpha-1-AT production segregated in the mouse-human hybrids concordantly with human purine nucleoside phosphorylase and with chromosome 14. All rat-human hybrids that were alpha-1-AT positive were also positive for human purine nucleoside phosphorylase and chromosome 14. Our study demonstrated the usefulness of rodent hepatoma cell hybrids for mapping human liver-specific genes because differentiated functions are expressed despite the fact that the human parental cells did not express these functions. Our study also showed that human alpha-1-AT gene product can be processed for secretion in the rodent hepatoma cellular environment. The mouse-human hybrids showed that no other human chromosome carries genes necessary for processing or secretion of human alpha-1-AT in the hybrid cell milieu.