Thirty-three rabbit cell lines were established from various fetal tissues of the inbred strain III of the New Zealand rabbit (Oryctolagus cuniculus). None of these lines exhibited senescence during a growth period of more than 2 years. Karyologic studies of most cell lines at 10 to 20 cell-passage intervals revealed that the karyotype stability of the rabbit cells in vitro was correlated with the organs from which the cell lines were derived. Thus, lines derived from cornea, spleen, and kidney tissues usually contained high frequencies of polyploidy in their early passages, whereas most of those derived from lung and skin were found to retain the normal diploid karyotype for much longer periods of time. One line derived from fetal lung tissue, designated Lung 16, remained diploid up to 100 passages. In late passages of the majority of all the lines studied, the cells became pseudodiploid, hyperdiploid, or polyploid. Among the pseudodiploid and the hyperdiploid cell lines, the chromosomal changes followed three basic patterns: (1) a gain of one or more telocentric chromosomes; (2) a loss of one telocentric chromosome plus a metacentric marker chromosome (M); or (3) a gain of a long telocentric marker chromosome with or without changes in the number of telocentric D chromosomes. By the G-banding technique, the telocentric chromosome involved in these three patterns was identified as the D-group chromosome 18 and the M marker chromosome as an isochromosome of 18. These results suggest that chromosomal rearrangement in rabbit cells involving trisomy of 18 may be responsible for the longevity of these cell lines cultured in vitro.