CHEF/18 fibroblastic cells derived from a Chinese hamster embryo are diploid and nontumorigenic and require multiple steps of chemical treatment and selection to produce tumorigenic derivatives. In this report, CHEF/18 cells and a mutant capable of growing in medium with a low concentration of serum, LS1-1, were recipients in DNA transfer experiments using the calcium phosphate coprecipitation method. Focus formation with donor DNAs from tumor-derived CHEF cells and from human bladder carcinoma cell line EJ gave yields of 0.02-0.59 focus per microgram of DNA per 10(6) recipients. In one experiment in which CHEF/18 cells were transfected with EJ DNA, the presence of human DNA was detected in five of seven foci by using a cloned Alu sequence. Cells from one of these foci gave rise to tumors in nude mice, and the DNA produced secondary CHEF/18 transfectants. Because normal human cells as well as CHEF/18 cells require multiple stages to become tumorigenic, these findings suggest that EJ cells contain tumor-inducing DNA as the result of prior changes that occurred during the development of this carcinoma.