A number of non-virally and non-chemically immortalized chicken embryo fibroblast (CEF) cells have been established recently in continuous cell culture. All immortal CEF cells tested showed common genetic alterations in the expression patterns of p53 and E2F-1 mRNA and protein which were down- and up-regulated, respectively. The biological effects of differentially regulated p53 and E2F-1 were determined by reporter gene transcriptional activity assays, DNA binding assays, and Northern blot analysis of the expression patterns of down-stream genes. In addition, expression of most of the cyclin genes was up-regulated in immortal CEF cells, which may be associated with the rapid cell division rates and serum-independent growth patterns seen in immortal CEF cells. The telomeric lengths and chromosome integrity were maintained in all immortal CEF cell lines without detectable telomerase activity. Although the functional inactivations of the p53 and Rb regulatory pathways are known to be common events for cellular immortalization, the genetic changes leading to alteration of p53 and E2F-1 function through transcriptional and post-transcriptional regulation seem to be unique in immortal CEF cells.