OBJECTIVES: The objective of this study was to evaluate the apparent diffusion coefficient (ADC) of diffusion-weighted magnetic resonance (MR) imaging for the differentiation of transition zone cancer from non-cancerous transition zone with and without prostatitis and for the differentiation of transition zone cancer Gleason grade (GG) using MR-guided biopsy specimens as a reference standard. MATERIALS AND METHODS: From consecutive MR-guided prostate biopsies (2008-2012) in our referral center, we retrospectively included patients from whom diffusion-weighted MR imaging ADC values were acquired during MR-guided biopsy and whose biopsy cores had a (cancer) core length 10 mm or greater and originated from the transition zone. Two radiologists, who were blinded to the ADC data, annotated regions of interest on biopsy sampling locations of MR-guided biopsy confirmation scans in consensus. Median ADC (mADC) of the regions of interest was related to histopathology outcome in MR-guided biopsy core specimens. Mixed model analysis was used to evaluate mADC differences between 7 histopathology categories predefined as MR-guided biopsy core specimens with primary and secondary GG 4-5 (I), primary GG 4-5 secondary GG 2-3 (II), primary GG 2-3 secondary GG 4-5 (III) and primary and secondary GG 2-3 cancer (IV), and noncancerous tissue without (V) or with degree 1 (VI) or degree 2 prostatitis (VII). Diagnostic accuracy was evaluated using areas under the receiver operating characteristic (AUC) curve. RESULTS: Fifty-two patients with 87 cancer-containing biopsy cores and 53 patients with 101 non-cancerous biopsy cores were included. Significant mean mADC differences were present between cancers (mean mADC, 0.77-0.86 Ãƒâ€” 10 mm/s) and noncancerous transition zone without (1.12 Ãƒâ€” 10 mm/s) and with degree 1 to 2 prostatitis (1.05-1.12 Ãƒâ€” 10 mm/s; P < 0.0001-0.05). Exceptions were mixed primary and secondary GG cancers versus a degree 2 of prostatitis (P = 0.06-0.09). No significant differences were found between subcategories of primary and secondary GG cancers (P = 0.17-0.91) and between a degree 1 and 2 prostatitis and non-cancerous transition zone without prostatitis (P = 0.48-0.94).The mADC had an AUC of 0.84 to differentiate cancer versus non-cancerous transition zone. AUCs of 0.84 and 0.56 were found for mADC to differentiate prostatitis from cancer and from non-cancerous transition zone. The mADC had an AUC of 0.62 to differentiate a primary GG 4 versus GG 3 cancer. CONCLUSIONS: The mADC values can differentiate transition zone cancer from non-cancerous transition zone and from a degree 1, and from most cases of a degree 2 prostatitis. However, because of substantial overlap, mADC has a moderate accuracy to differentiate between different primary and secondary GG subcategories and cannot be used to differentiate non-cancerous transition zone from degrees 1 to 2 of prostatitis. Diffusion-weighted imaging ADC may therefore contribute in the detection of transition zone cancers; however, as a single functional MR imaging technique, diffusion-weighted imaging has a moderate diagnostic accuracy in separating higher from lower GG transition zone cancers and in differentiating prostatitis from non-cancerous transition zone.