The prognostic value of Ki-67 expression, ploidy and s-phase fraction was evaluated in 133 patients with pancreatic cancer. Formalin-fixed paraffin-embedded surgical specimens of pancreatic ductal adenocarcinomas were stained with a polyclonal Ki-67 antibody. Ploidy and s-phase fraction was assessed by flow cytometry. The median percentage of Ki-67 positive nuclei was 26% (range 0-90%). The level of Ki-67 immunoreactivity was associated with TNM-stage, surgical resectability, tumour grade, ploidy and S-phase fraction. Ninetythree patients, with a nuclear expression of Ki-67 in < 50% of malignant cells, had a median survival of 12.8 months compared with 5.5 months for 25 patients with Ki-67 > or = 50% (p = 0.0008). Ploidy (p < 0.0001) and SPF (p = 0.002) were also significant prognostic factors in a univariate survival analysis. In a multivariate analysis, stage (or alternatively resectability), grade, ploidy and postoperative chemotherapy emerged as independent prognostic factors. When ploidy was excluded from the Cox multivariate model, S-phase fraction also predicted prognosis independently. Ploidy and S-phase fraction are independent prognostic factors in patients with pancreatic cancer. A high level of Ki-67 immunoreactivity is also an indicator of poor prognosis but seems to add little prognostic information to that provided by traditional parameters such as stage, resectability and grade.