Objective: To report MRI features of 3 cases with confirmed progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS) patients treated with natalizumab that presented with subtle new lesions on MRI several months prior to diagnosis. Background Clinical vigilance and early recognition of specific MRI changes are considered caveats for early diagnoses of PML in natalizumab treated patients. Design/Methods: Retrospective clinical and MRI evaluation of 3 patients with confirmed PML (by detection of JC virus in cerebrospinal fluid), who had several serial scans prior to the PML diagnosis. Results: Case 1: 41 year old man who received 29 monthly natalizumab infusions. A small new T2 hyperintense/T1 enhancing lesion in the subcortical white matter of the left frontal lobe was detected. No clinical symptoms were associated and he tested negative for prior JC virus exposure 6 months previous. Seven months later, a large T2 hyperintense/T1 enhancing lesion, indicative of PML was detected in the same location. He tested positive for JC virus exposure on repeat analysis, one month prior to diagnosis. Case 2: 47 year old woman on natalizumab for 31 months complained of new onset double vision. A small T2 hyperintense, T1 non-enhancing lesion was detected in pons. Two months later, a large T2 hyperintense lesion consistent with PML was detected in the same location, in addition to multiple other lesions. Case 3: 54 year old woman on natalizumab for 61 months presented with mild vertigo. A small new T2 hyperintense lesion in pons was seen retrospectively. Four months later a large T2 hyperintense lesion consistent with PML was detected in the same location, in addition to multiple other lesions. Conclusions: The appearance of subtle new T2 hyperintense lesions may suggest PML in patients treated with natalizumab for more than two years. Disclosure: Dr. Hojnacki has received personal compensation for activities with Biogen Idec, Serono, Inc., Pfizer Inc, and Teva Neuroscience as a speaker and/or participant on scientific advisory boards. Dr. Zivadinov has received personal compensation for activities with Teva Neuroscience, Biogen Idec, EMD Serono, and Questcor Pharmaceuticals as a speaker and/or consultant. Dr. Zivadinov has received research support from Biogen Idec, Teva Neuroscience, Genzyme Corporation, Bracco, Questcor Pharmaceuticals and EMD Serono. Dr. Weinstock-Guttman has received personal compensation for activities with Acorda Therapeutics, Biogen Idec, Serono, Inc., Novartis, Pfizer Inc, Teva Neuroscience as a speaker and/or participant on an advisory board. Dr. Weinstock-Guttman has received research support from Acorda Therapeutics, Biogen Idec, Serono Inc., Novartis, Pfizer Inc, Teva Neuroscience, National Multiple Sclerosis Society, the National Institutes of Health, ITN, Teva Neuroscience, Aspreva-Roche, Acorda Therapeutics and Shire Pharmaceuticals.