Objective: To design and perform a case-control study of multiple sclerosis (MS) in Crete, an island of 0.6 million people, that has experienced profound socioeconomic changes in recent decades. Methods: All MS cases occurring on Crete from 1980 to 2008 were ascertained. To search for putative risk factors, a structured questionnaire of 71 variables was employed, with patients with MS (n = 657) being compared to random controls (n = 593) matched for age, gender, and current place of residence. Results: MS incidence rose markedly on Crete over the past 3 decades. This increase was associated with a major shift in MS distribution among genders (1980: F/M = 0.9; 2008: F/M = 2.1), with females living in towns or having relocated at a young age from the countryside to urban centers being mainly affected. In rural Crete,MSshowed lesser increases and gender preference. Of the major changes that accompanied urbanization, smoking among women with MS increased dramatically, while imported pasteurized cow milk virtually replaced fresh goat milk produced locally. Compared to controls, female patients with MS more often used contraceptives and were older at first childbirth. Besides smoking, alcohol drinking and vitamin intake was more common among female patients with MS. Also, the distribution of childhood diseases and chronic medical conditions differed significantly between patients with MS and controls. Conclusions: MS incidence rose markedly over 3 decades in a genetically stable population in tandem with a transition from rural to urban living, thus possibly implicating environmental factors introduced by urbanization.