The frequency and timing of p53 inactivation in ulcerative colitis (UC)-associated tumorigenesis were investigated using immunohistochemistry (IHC) to detect p53 protein overexpression in 56 carcinomas and 40 dysplastic epithelia derived from 58 patients with UC undergoing colectomy for neoplasia. p53 DNA in 25 of the carcinomas also was evaluated by single-strand conformation polymorphism analysis (SSCP) to detect point mutations in exons 5-8 and by loss of heterozygosity analysis to detect allelic deletions. Point mutations were detected in 20 of the 25 carcinomas (80.0%) undergoing both IHC and DNA analysis. One carcinoma contained an allelic deletion but no mutations of the corresponding allele within the region tested. p53 overexpression occurred in 16 (76.2%) of the 21 carcinomas with point mutations and/or allelic deletions but not in any of those with wild type DNA. Of the 56 carcinomas evaluated by IHC, p53 overexpression occurred in 34 carcinomas (60.7%). The proportion of positive tumors was independent of stage, anatomic location, differentiation, and histological subtype. Overexpression was observed in nine of 20 dysplastic masses devoid of and situated remote from carcinoma (45.0%) and correlated positively with increasing grade of dysplasia (P < .025). In contrast, overexpression occurred in 16 of 20 dysplastic epithelia situated adjacent to carcinoma (80.0%) and correlated with overexpression by the corresponding carcinomas but not with the grade of dysplasia present (P = .013). It is concluded that p53 overexpression can be detected by IHC in most, although not all, UC-associated carcinomas with p53 mutations and/or allelic deletions. Based on this method, p53 overexpression occurs frequently in UC-associated carcinomas regardless of stage and pathological characteristics, in noncancerous dysplastic masses with high grade dysplasia, and in dysplasias of all grades situated adjacent to carcinomas. These findings implicate p53 inactivation in the progression from dysplasia to carcinoma in UC and suggest that its occurrence in dysplastic epithelium may be an independent marker of malignant potential.