OBJECTIVE Messenger ribonucleic acid for human glandular kallikrein (hK2), a protein similar to prostate-specific antigen (PSA), is expressed in the prostate. Quantitative tests for the relative amounts of PSA in serum have become important in the diagnosis and management of patients with prostate cancer. Measurement of hK2 in serum may also serve as a diagnostic indicator of disease. The object of this study was to determine if hK2 is present in the serum of patients with high serum concentrations of PSA. METHODS Recombinant prohK2 with an alanine to valine mutation at aa217 (phK2v217) was expressed in a hamster tumor cell line, AV12. The propeptide was treated with trypsin to yield the mature form of hK2 (hK2v217). Using a monoclonal antibody, HK1G586.1, which recognized wild type and mutant forms of pro- and mature hK2, an hK2-specific radioimmunoassay was developed. RESULTS PSA cross-reactivity in the radioimmunoassay (RIA) was 0.23%. hK2 was detected in the sera of 51 of 76 patients with PSA levels above 100 ng/mL. The dose-response curve of hK2-positive samples was linear, and recovery of phK2v217-spiked serum samples was close to 100%. The correlation between PSA and hK2 values in the patient sera was low (r = 0.168). CONCLUSIONS Given the importance of the role of PSA as a serologic indicator of prostate cancer, the demonstration that hK2 is also circulating in the blood of patients in different relative proportions to PSA suggests that it may be a significant novel marker for prostate cancer.