Although prostate-specific antigen (PSA) is a useful screening marker in prostate cancer, it has limited specificity. Previously it was shown that the amount of surface-bound PSA present on circulating macrophages was different between patients with localized prostate cancer and those with metastatic prostate cancer. It was recently demonstrated that intracellular PSA in macrophages can be measured by flow cytometry. In the context of searching for a noninvasive, highly reliable method for prostate cancer diagnosis, we assessed the extent to which extracellular (ie, surface-bound) and intracellular PSA-positive macrophages might differentiate patients with benign versus malignant prostatic disease. In a pilot study, the levels of complexed, surface-bound, and intracellular PSA were measured in 25 patients with elevated serum PSA values and histologically confirmed disease. In this group, no significant differences for serum PSA and complexed PSA levels, respectively, could be detected among patients with benign prostatic hyperplasia, prostatitis, and prostate cancer. Significant differences, however, were detected in intracellular PSA, although not in surface-bound PSA, among the 3 groups of patients. Intracellular PSA was measured prospectively in a second cohort of 189 patients who had a transrectal biopsy because of a serum PSA constellation suspicious for prostate cancer. In the expanded cohort, highly significant differences in intracellular PSA were observed between benign and malignant disease of the prostate, even in patients with serum PSA level<4 ng/mL. Screening of serum PSA alone or in combination with complexed PSA does not clearly distinguish patients with prostate cancer from those with prostatitis or benign prostatic hyperplasia. Macrophage intracellular PSA might represent a more.