AbstractSomatic cell hybridization is the method of choice to separate a chromosome of interest from the full chromosome complement and obtain a permanent source of the chromosome. This unit begins with the choice of fusion techniques and selectable markers for hybrids containing a chromosome of interest. The first set of protocols outline the production of whole‐cell hybrids by fusion of two cell lines: a monolayer (adherent) recipient and a donor that may be adherent or grown in suspension. The second set of protocols outline the production of micronuclei containing a limited number of chromosomes, and enucleation of the micronuclei to form microcells for fusion with recipient cells. Support protocols describe the preparation and use of cloning cylinders to isolate colonies in tissue culture, subcloning of whole‐cell hybrid populations to isolate lines that have segregated additional chromosomes, purification of microcell preparations, and molecular and cytogenetic methods for characterizing.