The principles and procedures of active specific immunotherapy developed from studies with the inbred guinea pig hepatocarcinoma model were used as the basis of a randomized, controlled prospective trial of active specific immunotherapy of colorectal cancer patients. The goal was to determine whether colorectal cancer patients treated with vaccines made of autologous tumor cells plus Bacillus Calmette-Guérin as adjuvant would have an increased reaction to their autologous tumor cells as measured by delayed-type cutaneous hypersensitivity (DCH) responses. Our results demonstrate that the active specific immunotherapy significantly increased the DCH responses to autologous tumor cells in 16 of 24 patients (67%). The DCH response of immunized patients to autologous normal mucosa, used as a normal tissue control, did not increase significantly. Furthermore, no significant DCH responses against autologous tumor or mucosa cells were detected in a group of nonimmunized control patients. The induced DCH responses were not correlated with other factors, such as the presence of bacteria in the cell preparation or the protein concentration of the cell preparations. The qualitative and quantitative differences in DCH responses to tumor cells and to normal mucosa cells suggest that the immunizations are targeted mainly to tumor-associated antigens with tissue-associated antigens playing a secondary role.