p53 gene alterations are among the most common events observed in colorectal cancer, and are accompanied frequently by DNA aneuploidy and high proliferative activity. The prognostic significance of such mutations remains controversial. We prospectively evaluated the prognostic significance of p53 mutations, DNA-ploidy, and S phase fraction (SPF) in a consecutive series of 160 colorectal cancer patients (median follow-up 71 months). Tumor DNA was screened for p53 mutations by PCR/single-strand conformational polymorphism/sequencing. DNA-ploidy and SPF were assessed by DNA flow cytometry. p53 mutations were detected in 68 of 160 (42.5%) cases. In 56% (38 of 68) of these, p53 mutations were found in conserved areas of the gene and in 44% (30 of 68 cases) outside the conserved regions. Eighteen of the 68 cases (26%) had mutations in the L3 loop, 11 of 68 (16%) in the L1 loop-sheet-α helix motif, and 39 of 68 (58%) outside L3 and loop-sheet-α helix. Seventy-five percent of the cases (120 of 160) showed DNA aneuploidy, whereas 18% of these (22 of 120) were multiclonal. The major independent predictors for both disease relapse and death were advanced Dukes' stage, p53 mutations affecting L3 loop, DNA-aneuploid tumors, and high SPF (>18.5%). Our results show that mutations in L3 functional domain, more than any mutations, are important biological indicators to predict the outcome of patients indicating that these mutations have biological relevance in terms of colorectal cancer disease course.