Polyethylene glycol (PEG)-mediated cell fusion is a simple and efficient technique used widely for the production of somatic cell hybrids and for nuclear transfer in mammalian cloning. We describe a basic protocol of PEG-mediated cell fusion for the production of somatic cell hybrids. Fusion can be performed between adherent and suspension cells or between adherent cells or suspension cells. Either whole cells or microcells can be used as donors to fuse with recipient cells. Microcell fusion is particularly useful in transfer of a single or a limited number of chromosomes between various types of cells. Using this method, we have successfully introduced mammalian minichromosomes into a variety of vertebrate cells. The technique described here can be adapted for uses in other cell fusion involved research. This protocol, in principle, provides guidelines for further development of PEG mediated cell fusion technology.