Five clones of Chinese hamster cells transformed with simian virus 40 (SV40) were isolated from methylcellulose and characterized as to Giemsa-banded karyotype, DNA content, saturation density, agglutination with concanavalin A, and tumorigenicity. Chromosome analysis and DNA content studies at early passage revealed that the genetic complement for all clones was predominantly near tetraploid. All cultures examined contained a proportion of hypertetraploid cells. Nonrandom chromosome changes included at least one broken No 1 chromosone in 80% or more of the cells in each clone, and fewer sex chromosomes than anticipated from the ploidy of the cells. Several abnormal marker chromosomes tended to recur. These changes were more pronounced in the cells cultured from tumors formed by three of the clones. A karyotypically stable stem line was not noted for any of the clones or tumors. The functional significance of the karyotypic heterogeneity was assessed by means of cloning efficiencies both on plastic and in methylcellulose.