PEG-mediated fusion between mouse Cl1d cells and primary Chinese hamster spleen cells produced interspecific hybrids which slowly and nonrandomly segregated Chinese hamster chromosomes. Cytogenetic and isozyme analysis (31 loci) of HAT and BrdU selected hybrid clones and subclones and of members of a hybrid clone panel retaining different combinations of Chinese hamster chromosomes enabled provisional assignment of the following enzyme loci on Chinese hamster chromosomes: thymidine kinase, galactokinase, and acid phosphatase-1 to chromosome 7; galactose-1-phosphate uridyltransferase to chromosome 2; and adenosine kinase, esterase D, glutathione reductase, glyoxalase, nucleoside phosphorylase, peptidases B and S, and phosphoglucomutase (PGM) 2 to chromosome 1. Assignments of PGM1, 6-phosphogluconate dehydrogenase, and enolase 1 to chromosome 2 were confirmed, and a chromosome 2 deletion (q23-q33) enabled the provisional assignment of PGM1 to that region. The assignments provide markers for the study of the genetic consequences of chromosomal rearrangements in Chinese hamster cell lines and support the concept of conservation of mammalian autosomal linkage groups.