Squamous cell carcinoma (SCC) immortality is associated with p53 and INK4A dysfunction, high levels of telomerase and loss of heterozygosity (LOH) of other chromosomes, including chromosome 4. To test for a functional cancer mortality gene on human chromosome 4 we introduced a complete or fragmented copy of the chromosome into SCC lines by microcell-mediated chromosome transfer (MMCT). Human chromosome 4 caused a delayed crisis, specifically in SCC lines with LOH on chromosome 4, but chromosomes 3, 6, 11 and 15 were without effect. The introduction of the telomerase reverse transcriptase into the target lines extended the average telomere terminal fragment length but did not affect the frequency of mortal hybrids following MMCT of chromosome 4. Furthermore, telomerase activity was still present in hybrids displaying the mortal phenotype. The MMCT of chromosomal fragments into BICR6 mapped the mortality gene to between the centromere and 4q23. Deletion analysis of the introduced chromosome in immortal segregants narrowed the candidate interval to 2.7 Mb spanning D4S423 and D4S1557. The results suggest the existence of a gene on human chromosome 4 whose dysfunction contributes to the continuous proliferation of SCC and that this gene operates independently from telomeres, p53 and INK4A.