Objective <p>This study aims to investigate the efficacy and safety of prostate-specific membrane antigen (PSMA) radiolabeled with copper-64 (⁶⁴Cu) using the bifunctional chelating agents (BFCAs) NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). As widely utilized BFCAs in the development of radiopharmaceuticals, NOTA and DOTA play a critical role in ensuring stable chelation with ⁶⁴Cu. This study evaluates the stability, bioavailability, and therapeutic potential of these radiolabeled compounds in preclinical models and initial clinical trials.</p> Methods <p>⁶⁴Cu-DOTA-PSMA-3Q and ⁶⁴Cu-NOTA-PSMA-3Q were synthesized by manual labeling. The radiochemical purity, stability, specificity and biological distribution of the product were evaluated by preclinical studies. In 23 patients with suspected prostate cancer, PET/CT imaging was used to evaluate the potential and differences in biological distribution of ⁶⁴Cu-DOTA-PSMA-3Q and ⁶⁴Cu-NOTA-PSMA-3Q in clinical diagnosis.</p> Results <p>The radiochemical purities of ⁶⁴Cu-DOTA-PSMA-3Q and ⁶⁴Cu-NOTA-PSMA-3Q are more than 98% and have good stability in vitro. Biodistribution studies in healthy mice revealed that both tracers primarily underwent renal excretion post-injection. Liver uptake of ⁶⁴Cu-DOTA-PSMA-3Q was significantly higher than that of ⁶⁴Cu-NOTA-PSMA-3Q at 1&#xa0;h after injection (<i>P</i>&lt;0.05). Micro-PET/CT imaging in 22Rv1 tumor-bearing mice demonstrated similar tumor uptake for both tracers at 1&#xa0;h after injection (<i>P</i>&gt;0.05). However, after 24&#xa0;h, ⁶⁴Cu-DOTA-PSMA-3Q exhibited significantly better tumor retention compared to ⁶⁴Cu-NOTA-PSMA-3Q (<i>P</i>&lt;0.05). In clinical PET/CT imaging involving 23 patients with suspected prostate cancer, no adverse reactions or significant changes in vital signs were observed, underscoring the safety of both tracers. Notably, ⁶⁴Cu-NOTA-PSMA-3Q demonstrated higher uptake in the lacrimal glands (17.73 vs. 10.84), parotid glands (20.98 vs. 16.30), and submandibular glands (20.26 vs. 17.28) compared to ⁶⁴Cu-DOTA-PSMA-3Q. Conversely, uptake in the sublingual glands was lower for ⁶⁴Cu-NOTA-PSMA-3Q (7.10 vs. 7.49). Of particular clinical relevance, liver uptake of ⁶⁴Cu-NOTA-PSMA-3Q was significantly lower than that of ⁶⁴Cu-DOTA-PSMA-3Q (4.04 vs. 8.18), highlighting a key difference in their biodistribution profiles.</p> Conclusions <p>Both NOTA and DOTA are suitable chelators for the development of ⁶⁴Cu-labeled PSMA-3Q tracers for PET/CT imaging. DOTA showed better tumor retention 24&#xa0;h after injection, while NOTA showed lower uptake in the liver, in addition, NOTA was higher uptake in the salivary glands, while DOTA was lower uptake in these tissues. Overall, these findings highlight the importance of selecting the right chelating agent to optimize clinical imaging outcomes.</p> Trial registration <p>Chinese Clinical Trial Registry ChiCTR2300072655, Registered 20 June 2023.</p>