Mutated p21 ras proteins contain single substituted amino acid residues and represent cancer-specific proteins. The current study examined whether primed T cell immunity to mutant p21 ras proteins and/or peptides can be detected in patients with pancreatic or colon cancer. Studies focused on the aspartic acid substitution in amino acid position 12 (denoted D12) as the commonest mutation in gastrointestinal malignancy. Peripheral blood lymphocytes from patients or normal individuals were tested for the ability to proliferate in response to normal or mutated ras peptides or proteins. T-cell responses were defined as a stimulation index of > 2.0. Results showed that 7 of 16 (44%) pancreatic cancer patients responded to ras-D12 peptide. Responses to ras-D12 protein were studied in only the last four patients that responded to D12 peptides. Three of the 4 patients that responded to ras-D12 peptide showed a substantial response to p21 ras-D12 protein (stimulation indices of 12, 8, and 24). Specificity was validated by examining responses to normal and alternate ras peptides and proteins. T-cell responses to ras-D12 peptides were detected in only 2 of 25 (8%) colon cancer patients. None of 11 normal individuals tested had positive responses to normal or mutant ras p21 proteins and/or peptides. Thus, CD4+ T-cell immunity to the mutated segment of ras protein is present in some patients with gastrointestinal cancer.