Purpose To determine the impact of three different methods of region of interest (ROI) positioning for apparent diffusion coefficient (ADC) measurements on the assessment of complete response (CR) to neoadjuvant combined chemotherapy and radiation therapy (CRT) in patients with rectal cancer. Materials and Methods Institutional review board approval was obtained for this study; all patients gave written informed consent. ADCs were measured by two radiologists using three circular ROIs (three-ROIs), single-section (SS), and whole-tumor volume (WTV) methods in 62 patients with locally advanced rectal cancer on pre- and post-CRT images. Interobserver variability was analyzed by calculating intraclass correlation coefficient (ICC). Descriptive statistics and areas under the receiver operating characteristic curves (AUCs) were calculated to evaluate performance in determining CR from pre- and post-CRT ADCs and ADC change. Histopathologic tumor regression grade was the reference standard. Results SS and WTV methods yielded higher AUCs than did the three-ROIs method when determining CR from post-CRT ADC (0.874 [95% confidence interval {CI}: 0.778, 0.970] and 0.886 [95% CI: 0.781, 0.990] vs 0.731 [95% CI: 0.583, 0.878], respectively; P = .033 and P = .003) and numeric change (0.892 [95% CI: 0.812, 0.972] and 0.897 [95% CI: 0.801, 0.994] vs 0.740 [95% CI: 0.591, 0.890], respectively; P = .048 and P = .0021). Respective accuracies of SS, WTV, and three-ROIs methods were 79% (49 of 62), 77% (48 of 62), and 61% (38 of 62) for post-CRT, 79% (49 of 62), 86% (53 of 62), and 60% (37 of 62) for numeric ADC change, and 77% (48 of 62), 84% (52 of 62), and 57% (35 of 62) for percentage ADC change (ADC cut-offs: 1.21, 1.30, and 1.05 × 10-3 mm2/sec, 0.33, 0.45, and 0.27 × 10-3 mm2/sec increases, and 40%, 54%, and 27% increases, respectively). Post-CRT and ADC change measurements achieved negative predictive values of 96% (44 of 46) to 100% (39 of 39). Intraobserver agreement was highest for WTV-derived ADCs (ICC, 0.742 [95% CI: 0.316, 0.892] to 0.891 [95% CI: 0.615, 0.956]) and higher for all pretreatment than posttreatment measurements (ICC, 0.761 [95% CI: 0.209, 0.930] and 0.648 [95% CI: 0.164, 0.895] for three-ROIs method, 0.608 [95% CI: 0.287, 0.844] and 0.582 [95% CI: 0.176, 0.870] for SS method, 0.891 [95% CI: 0.615, 0.956] and 0.742 for WTV method [95% CI: 0.316, 0.892]). Conclusion Tumor ADCs are highly dependent on the ROI positioning method used. Larger area measurements yield greater accuracy in response assessment. Post-CRT ADCs and values of ADC changes accurately identify noncomplete responders. WTV measurement of percentage ADC change provides the best results. © RSNA, 2016 An earlier incorrect version of this article appeared online. This article was corrected on September 19, 2016.