We have studied the kinetics with which cultures of primary mouse embryo cells pass through the crisis period, escape their terminal differentiation (cellular senescence), and give rise to an immortal cell line. The process is strain-dependent, with cells from the outbred Swiss CD-1 mouse being considerably more adept at forming an immortal 3T3 line than cells from the inbred SWR line; Balb/c cells appeared intermediate in their behavior. The continued presence of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate or the poly(ADPribose)polymerase inhibitor 3-aminobenzamide affected the kinetics but did not seem to alter the outcome. Changes in expression of various genes, including those encoding mitogen-regulated protein (proliferin), endogenous gag-pol retrovirus sequences, insulin-like growth factor II, and a variety of protooncogenes, were monitored during the process of immortalization, and although certain changes were reproducibly characteristic of cells from a given mouse strain passed according to a specific regimen, none of the observed changes were reproducibly characteristic under all conditions of immortalization. In particular, our data indicate the absence of a strict correlation between cellular immortalization and the activation of endogenous gag-pol expression. We conclude from our observations that the establishment of permanent lines from primary mouse embryo cells in serum-containing medium reflects the selection of a variant subpopulation of cells that did not preexist but rather arose in response to the specific culture conditions by a process resembling differentiation. Multiple and complex changes in gene expression occur that are affected by the culture conditions and the strain (genotype) of the mouse.