Background <p>Several factors predict the detection rate of ¹¹C-choline PET/CT in prostate cancer (PCa) patients. The role of the Gleason score (GS) is disputed.</p> Objective <p>To assess whether GS predicts ¹¹C-choline PET/CT in PCa patients.</p> Methods <p>520 PCa patients with biochemical failure after radical prostatectomy were retrospectively recruited. Univariate and multivariate binary regressions analysis was used to assess the role of predictive factors.</p> Results <p>Patients with GS &gt; 7 had significantly (P &lt; 0.05) greater number of positive ¹¹C-choline PET/CT than both patients with GS &lt; 7 and patients with GS = 7. ¹¹C-choline PET/CT positive detection rate in patients with GS = 7 did not significantly differ from patients with GS &lt; 7 (P &gt; 0.05). The predictive effect of GS could be detected only in patients never treated with anti-androgen therapy (ADT). Patients with GS &gt; 7 had, in comparison to both patients with GS &lt; 7 and to patients with GS = 7, significantly (P &lt; 0.05) higher number of pathological ¹¹C-choline uptake sites in pelvic or retroperitoneal lymph nodes, and in the skeleton, but not in the prostatectomy bed. However, at multivariate analysis, statistical significance was preserved by age, pathological stage and biochemical failure during ADT, but not by GS.</p> Conclusion <p>Only GS &gt; 7 predicted positive ¹¹C-choline PET/CT and this effect was limited to drug-free and drug-naïve PCa patients, and to univariate analysis. The power of GS to predict positive ¹¹C-choline PET/CT is lower than that of other biological factors. Methodological and biological factors affecting these results are discussed.</p>