Many p53 functions require p53 transport into the nucleus. Mutant p53 also generally accumulates in the nucleus of transformed or neoplastic cells. However, examples of cytoplasmic accumulation of wild-type or mutant p53 have also been reported. Various explanations have been provided for defective nuclear localization. Here we propose a novel example of cytoplasmic p53 localization which occurs in cells showing gene amplification and appears to be due to the formation of stable p53 multimers. We studied a methotrexate-resistant Chinese hamster cell line (MTX M) carrying amplified dihydrofolate reductase genes and derived from a cell line with p53 nuclear accumulation. MTX M showed cytoplasmic p53 localization and, on immunoblots, several extra bands in the high molecular weight region, besides the expected 53 kDa band. p53 localization and the appearance of high molecular weight bands appeared to be correlated with the degree of DNA amplification. However, amplification of dihydrofolate reductase itself was not involved. Changing the p53 phosphorylation status quantitatively influenced the formation of high molecular weight bands. Cell fusion experiments demonstrated that p53 cytoplasmic localization in MTX M is a dominant phenotype. This result suggests that the defect causing lack of nuclear localization in this cell line does not reside in the nucleus. In the cytoplasm of MTX M and of wild-type/MTX M heterodikaryons p53 gives rise to protein complexes that are unable to re-enter the nucleus. The formation of such protein complexes is dependent on the amplification of an unknown gene product.