Clones of SV40 tsA mutant-transformed mouse embryo cells with a temperature-independent transformed growth phenotype were derived from a parental line with a temperature-dependent growth phenotype, using the selective pressure of anchorage independent growth in agar at 40 degrees C. The parental J78 clone contained a complex tandem structure of integrated SV40 DNA within two cellular DNA fragments of 10 and 14 kb, generated by Bgl II digestion. The new clonal derivatives, in addition to an altered growth phenotype, displayed additional high molecular weight sites of SV40 DNA integration. A marked structural similarity among integration sites suggested that the new sites may have arisen by duplication and translocation of an original integration structure, or via an unequal crossover event. The rearrangement of viral DNA sequences appears to be specifically associated with the emergence of temperature-independent clones, since the isolation of new clones under nonselective conditions, that is, growth in semi-solid medium at 33 degrees C, was not accompanied by modification in cellular sequences containing SV40 DNA.