We analyzed the p53 mutational status, mRNA and protein expression in 24 human breast carcinoma cell lines. Following measurement of their DNA content with flow cytometry, we ascertained the copy numbers of the centromere of chromosome 17 (cen17) and p53 with fluorescence in situ hybridization (FISH). A functional yeast assay (FASAY) was used to screen for inactivating mutations. Positive results were subsequently verified by DNA sequencing. Finally, we assessed the mRNA expression with a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay and the protein expression with immunocytochemical staining, western blot, and quantitative flow cytometry. The DNA content of the cell lines ranged from 0.85 to 2.58. Nine cell lines had concordant copy numbers (between two and four) of p53 and cen17, whereas 12 had more, and three less cen17 than p53 copies. The FASAY was successful in all but one cell line and revealed the presence of mutated alleles in 16 of them, 13 cell lines expressed only the mutated, and three both the mutated and the wild-type alleles. The mutations were comprised of 11 missense, two nonsense, and three frameshift mutations. Immunocytochemical staining, western blot and quantitative flow cytometry yielded comparable p53 protein expression results. However, both the mRNA and the protein expression levels varied considerably in the different cell lines and no consistent pattern with regard to the respective p53 mutational status became evident. The results obtained in these breast carcinoma cell lines indicate that no clear-cut linear relationship exists between the p53 mutational status and the extent of its respective mRNA and protein expression. Therefore, direct DNA analyses and functional assays remain the only methods for the reliable detection of p53 mutations.