Results from ecological, case-control and cohort studies have shown that vitamin D reduces the risk of bone fracture, falls, autoimmune diseases, type 2 diabetes, CVD and cancer. However, there is still epidemic vitamin D insufficiency especially among individuals living at high latitudes or with dark skin. Serum levels of 25-hydroxyvitamin D (25(OH)D) are considered the best biomarker of vitamin D nutritional status. Appropriate sunshine exposure or oral supplementation is necessary to maintain sufficient vitamin D status, which is generally accepted as serum 25(OH)D>75 nmol/l. Immunoassays, especially RIA, have been primarily used to measure serum 25(OH)D while liquid chromatography-MS (LC-MS) is considered the 'gold standard'. There is significant disparity among the immunoassays, and all immunoassays have considerable bias compared with LC-MS methods. Because of the variations among the results from these different assays, it is necessary that assay-specific reference ranges be established or standardisation of the assays take place. The present review focuses on ecological, case-control, and cohort studies that investigated the role of vitamin D in health and disease. In addition, analytical techniques used in laboratory evaluation of vitamin D nutritional status are also critically reviewed. The majority of the literature included in the present review is selected from that searchable in PubMed up to the end of September 2008.