It has been suggested that natalizumab-associated progressive multifocal leukoencephalopathy may be prevented by structured interruptions of treatment. Evidence supporting such a drug holiday is not yet available. Here we present initial observations in 10 multiple sclerosis patients who were stringently monitored up to 6 months after discontinuation of the infusions. Cumulatively, a combination of clinical relapse and new and/or enhanced lesions on magnetic resonance imaging had occurred in 7 of 10 patients. Although numbers are small, our data suggest that in patients who were switched to natalizumab because of disease activity despite first-line treatment, a natalizumab drug holiday without reinstatement of alternate disease-modifying therapy is poorly tolerated.