The transcription of the ΔN133p53 isoform of the TP53 gene is controlled by an internal promoter region (IPR) containing eight polymorphisms in 11 common haplotypes, following a resequencing of 47 Caucasians. We assayed the functional effects of the commonest six haplotypes on the promoter activity with a luciferase reporter system, in HeLa and 293T cells. These studies showed that different IPR haplotypes are associated with differences in the promoter activity resulting in marked variation in the baseline expression of ΔN133p53. In vivo quantitative-polymerase chain reaction (PCR) on human tissues confirmed that the baseline levels of ΔN133p53 showed haplotype specific differences that paralleled those seen in vitro. When cell lines were treated with camptothecin, the fold-increase in ΔN133p53 levels was dose-dependent but haplotype-independent (i.e., similar for all the haplotypes). Finally, we used an electrophoretic mobility shift assay to analyze the rs1794287 polymorphism and found changes in the pattern of protein binding. This partially confirmed our in silico analysis showing that the polymorphism rs1794287 can affect the function of the internal promoter by changing its affinity for several transcription factors. Thus, we showed that the expression of ΔN133p53 is under genetic control, and suggested the presence of interindividual differences underlying this mechanism. Hum Mutat 31:1–10, 2010. © 2010 Wiley-Liss, Inc.