PSA is the most common biomarker used in the screening and monitoring of prostate cancer. However, changes in PSA do not always reflect disease dynamics in every patient, and antihormonal agents may modulate its levels without significant antitumor effects. Changes in circulating tumor cells (CTC) have been described as a more objective measure of treatment response. Differences between PSA and CTC may be explained by heterogeneity in tumor cells producing PSA. To explore this, we measured the PSA secretion from a single CTC to gain insights into the PSA secretion heterogeneity between tumor cells. CTCs were enriched using EpCAM-based immunomagnetic enrichment in diagnostic leukapheresis of 18 patients with metastatic castration-naïve prostate cancer (mCNPC) not pretreated with any therapy, including androgen deprivation therapy. Calcein+ CD45- cells were sorted by flow cytometry and deposited as single cells on a nanowell array to measure the PSA secretion after 24 hours. In nine of 18 patients, PSA secretion was detectable and observed from both prostate-specific membrane antigen-positive and prostate-specific membrane antigen-negative CTCs. In these patients, 29% to 100% (mean, 52; median, 47) of CTCs secreted PSA, with average PSA secretion levels ranging from 4 to 11.68 pg/cell (mean, 6.38 ± 2.29; median, 6.05). Notably, a strong heterogeneity in PSA secretion was observed within each patient. Our study demonstrates that CTC in mCNPC, even before therapy, produces varying amounts of PSA and often no PSA. These findings may explain the shortcomings of PSA as a biomarker for therapy response. SIGNIFICANCE: This study reveals heterogeneity in PSA secretion among individual CTCs from patients with mCNPC prior to any therapeutic intervention, thereby highlighting the limitations of PSA as a biomarker.