A major question in carcinogenesis is, How can a normal cell accumulate multiple mutations in different genes on different chromosomes, when the mutation rate of each gene is in the range of 10-8 to The strategy used in the present investigation is to search directly for the existence of second-site mutations in the TK6 human lymphocyte cell line by screening for changes in a number of PCR microsatellite markers located throughout the genome in a set of independent cell clones selected for new mutations at the thymidine kinase (TK) gene. Both normal and slow-growth mutants were examined (36). A set of randomly chosen control cell clones not selected for TK mutations was also examined to establish a background frequency of mutations at these microsatellite loci. Our hypothesis would predict that the frequency of mutation at the microsatellite loci should be significantly higher in cells selected for mutations at TK than in nonselected control clones. MATERIALS AND METHODSCell culture and mutant selection. The methods for the growth and maintenance of TK6 human lymphoblastoid cells have been described previously (10, 21). Briefly, they were maintained in suspension culture at 37°C in a humidified 5% CO2 atmosphere in RPMI 1640 growth medium supplemented with 10% heat-inactivated horse serum and 1% penicillinstreptomycin (Gibco Laboratories, Grand Island, N.Y.