Chicken fibroblast cells isolated from 7-day-old White Leghorn S-line chicken embryos were used for microcell hybridization. Cells transfected with a pSV2-neo plasmid, which carries the Neor gene and confers resistance to geneticin (G418), were selected and maintained in medium containing G418 (350 micrograms/mL). Cells were micronucleated by colcemid arrest and hypotonic treatment. Enucleation was carried out by centrifugation in a Percoll gradient in the presence of cytochalasin B. Purified microcells and HeLa S3 cells were mixed and agglutinated by addition of phytohemagglutinin P, followed by polyethylene glycol fusion to generate microcell hybrids. Chicken by human microcell hybrids were selected in RPMI-1640 medium containing 1.4 mg/mL G418. Cloned hybrid cell lines were maintained in the same medium containing .7 mg/mL of G418. The presence of chicken chromosomes in hybrid cells was demonstrated by cytogenetic analysis and high-resolution nonisotopic chromosomal in situ hybridization. Three out of 28 hybrid cell lines analyzed retained single chicken chromosomes.