Levels of serum antibody to Bacillus Calmette-Guérin (BCG) were determined by solid-phase radioimmunoassays in 48 normal donors and 60 patients with bladder cancer. Of 57 patients enrolled in a randomized prospective controlled trial of BCG immunotherapy, 47 were followed for up to 30 months, thus permitting comparisons of tumor recurrence, delayed cutaneous hypersensitivity responses to purified protein derivative (PPD), and serum BCG antibody levels at specific intervals during the clinical course. Sera from normal donors and cancer patients prior to BCG therapy had equally low levels of BCG antibody. AFter administration of intravesical and percutaneous BCG, significant rises of serum BCG antibody levels were detected in 23 of 24 randomized BCG immunotherapy patients. Skin test responses to PPD and serum BCG antibody levels had a close correlation as immune response indicators in 14 of 24 BCG therapy patients, while rises in serum BCG antibody levels were a better response indicator than PPD skin test reactions in the other 10 patients. Eleven of the 23 patients randomized into the non-BCG treatment group had tumor recurrence, although tumors recurred in only six of the 24 randomized patients in the BCG therapy group. Two additional nonrandomized BCG-treated patients had tumor recurrence. All eight BCG-treated patients with tumor recurrence had documented increases in serum BCG antibody levels after BCG therapy. Only three of these eight patients had conversion of PPD skin test responses from negative to positive; three were positive before immunotherapy and two remained negative after BCG therapy. Levels of antibodies reactive with human adenovirus type 5 and with Escherichia coli antigens were similar in sera from normal donors and from the randomized bladder cancer patients in both the BCG and non-BCG treatment groups. These results suggest that serum BCG antibody responses are as useful as PPD skin tests in identifying immunological responses to the immunoadjuvant BCG during immunotherapy trials in cancer patients.