Background: Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancers, making it an ideal imaging and therapeutic target of interest. The utility of the PSMA-targeted imaging agent ^99mTc-EC0652 is being evaluated, along with biomarker analysis of circulating tumor cells (CTCs), in pts with mCRPC in a PSMA-targeted chemotherapeutic study. We now report the PSMA heterogeneity via CTC vs. imaging in the pt population treated to date. Methods: Patients were enrolled in 1 of 2 cohorts: mCRPC taxane naïve or taxane exposed. A total of 48 pts evaluated at the time of the data cut had baseline CT & bone scans performed in addition to a ^99mTc-EC0652 SPECT/CT as a measure of imaging-based PSMA expression. 40 of 48 pts provided pre-treatment blood samples evaluable for CTC biomarker analysis, which included PSMA expression & were given percent homologous recombination deficiency (%HRD) scores. Four of the best and four of the worst responders were evaluated in more detail for imaging and CTC-based biomarker analysis as it related to clinical outcome. Results: 35 of 40 pts (88%) had detectable CTCs in their samples. 15 of the 35 (43%) pts had PSMA-positive CTCs. The 4 “non-responders” were on study for an average of 41 days and the 4 “responders” for an average of 256 days. Each group contained 2 taxane exposed & 2 taxane naïve patients. Of the 335 bone lesions analyzed by MDP, CT, ^99mTc-EC0652, 333 (99.4%) were characterized as PSMA_pos based on their ^99mTc-EC0652 SPECT/CT scan. Of the 26 soft tissue lesions analyzed by CT, all were characterized as PSMA_pos based on their ^99mTc-EC0652 SPECT/CT scan. Seven (26.9%) of those lesions were CT positive (observed on CT scan). Each group had 2 pts that were CTC-based PSMA_pos& 2 that were PSMA_neg. Their percent homologous recombination deficiency (%HRD) scores were of 0.60 [0.333, 1.0] for “non-responders”& 0.01 [0.0, 0.039] for “responders”. Conclusions: PSMA-based imaging showed a high percentage of positive lesions whereas CTC-based PMSA positivity is lower by comparison (43%). The discordance between the imaging results & CTC-based biomarkers, & the relative therapeutic predictive value, requires additional exploration. Clinical trial information: NCT02202447