Background: The purpose of this prospective, nonrandomized, pilot imaging study (NCT02420977) is to evaluate prostate-specific membrane antigen (PSMA), [18F]DCFPyL (DCFPyL), as a PET/CT imaging biomarker of androgen receptor (AR) signaling in newly diagnosed PCa. Methods: 23 men with treatment-naïve, newly diagnosed high-risk prostate cancer were enrolled. Patients underwent DCFPyL PET/CT imaging and MRI/ultrasound fusion-guided prostate biopsy before and after 2-3 months of nADT (LHRH agonist and anti-androgen), followed by definitive IMRT. We measured the total number of lesions, lesion location, maximum standardized tumor uptake value (SUVmax), PSA, and changes in those variables between baseline and follow-up scans. Concordance between biopsy pathology results and PSMA imaging parameters before and after ADT was evaluated. Results: 3 patients were excluded after baseline PET/CT due to high-burden metastases, small cell histology, and declining to proceed further. 20 patients remained for evaluation. At baseline, 19/20 and 8/20 had at least 1 avid intraprostatic and extraprostatic lesion, respectively, with a 50 total lesions (28 prostate, 19 nodal, 3 bone); median intraprostatic and extraprostatic SUVmax were respectively 9.6 (range: 3.3-56.8) and 7.0 (range: 2.3-61.9). Post-nADT scans revealed 32 lesions (19 prostate, 11 nodes, 2 bone); median intraprostatic and extraprostatic SUVmax decreased respectively to 4.1 (range 1.9-27.8) and 3.2 (range: 1.7-8.9). 3/19 (15.8%) of the patients demonstrated complete uptake resolution of the avid intraprostatic foci, 15/19 (78.9%) showed decreased uptake (58.3% median decrease in SUVmax), and 1/19 (5.26%) showed increased SUVmax. Of the 8 patients with extraprostatic lesions, 2/8 (25%) demonstrated complete uptake resolution, 5/8 (62.5%) showed partially decreased uptake (66.67% median decrease in SUVmax), and 1/8 (12.5%) showed increased SUVmax. One of the same patients still had 2 lesions, for a total of 13 intraprostatic target lesions. Regarding concordance between pathologic results and PSMA imaging response pre- and post-nADT, 14/19 (73%) had biopsies fully concordant with PSMA scan (i.e. residual tumor and residual PET avidity), 4/19 (21%) had biopsies mostly concordant with the scan (i.e. complete pathologic response with partial PET response or vice versa), and 1/19 (5%) had a biopsy discordant with the PSMA scan (i.e. no pathology response, but complete response on PSMA). Conclusions: Post-nADT PET detected fewer PSMA-positive lesions with overall decreased or resolved tumor uptake, except for 2 separate patients with increased intra- and extraprostatic uptake. 95% of post-nADT PSMA scans were fully or mostly concordant with post-nADT biopsies, suggesting that PSMA scans may be a clinically useful, prognostic imaging biomarker for disease status evaluation post-nADT. Clinical trial information: NCT02420977.