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To quantify variations within and between observers ascribable to manual region of interest (ROI) placement in patients with breast cancer undergoing dynamic MRI.Expert and nonexpert observers independently outlined tumor ROIs on 30 dynamic T1-weighted (T1W) MRI scans on five occasions over two months. Lesion size (number of pixels) and kinetic parameter estimates, including the transfer constant (Ktrans), were calculated for each ROI placement. Inter- and intraobserver variability was assessed with respect to the interval between drawings, lesion morphology, and observer experience.For the nonexpert, the variability reduced with decreasing time intervals between ROI drawings (the coefficient of variance (wCV) values at two months, two weeks, one day, and same-day time intervals were respectively 11.6%, 10.7%, 4.8%, and 2.6% for lesion size, and 8.9%, 9.7%, 6.7%, and 3.2% for Ktrans). For the expert observer, the variability was smaller overall and more constant, but improved for same-day ROI placements (region size wCV: 7.5%, 6.2%, 7.1%, and 3.7%; Ktrans wCV: 5.4%, 5.3%, 5.6%, and 4.5%).Significant observer variability in manual ROI placement occurs in dynamic MRI of breast cancer. For serial patient studies, ROI placements should be outlined at the same sitting to minimize observer error.