For nearly 60 years, the Framingham Heart Study has examined the natural history, risk factors, and prognosis of cardiovascular, lung, and other diseases. Recruitment of the Original Cohort began in 1948. Twenty-three years later, 3,548 children of the Original Cohort, along with 1,576 of their spouses, enrolled in the Offspring Cohort. Beginning in 2002, 4,095 adults having at least one parent in the Offspring Cohort enrolled in the Third Generation Cohort, along with 103 parents of Third Generation Cohort participants who were not previously enrolled in the Offspring Cohort. The objective of new recruitment was to complement phenotypic and genotypic information obtained from prior generations, with priority assigned to larger families. From a pool of 6,553 eligible individuals, 1,912 men and 2,183 women consented and attended the first examination (mean age: 40 (standard deviation: 9) years; range: 19–72 years). The examination included clinical and laboratory assessments of vascular risk factors and imaging for subclinical atherosclerosis, as well as assessment of cardiac structure and function. The comparison of Third Generation Cohort data with measures previously collected from the first two generations will facilitate investigations of genetic and environmental risk factors for subclinical and overt diseases, with a focus on cardiovascular and lung disorders.