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Objectives: Most comparisons of health in Europe take place at the national level. However, there is increased interest in looking at health data at a sub-national level. This study aimed to establish the availability of health data at a regional level and to develop a methodology for the comparisons of health indicators at a sub-national level. In previous work on indicators at a regional level in the European Union, the authors recommended the development of such indicators. This paper takes into account the expansion of the European Union which took place in 2004. Study design: Observational study using routinely available data. Methods: Similar to previous projects, a network of country correspondents was used and data were collected on a similar range of topics. In addition, a supplementary list of data was collected from one region of each country. Results: Twenty-three countries out of the 25 member states of the European Union participated in the study. Where available, data were of relatively good quality. Data on mortality were most readily available, but data on important public health topics such as obesity were much more difficult to obtain. Conclusions: A database and a set of indicators for relevant sub-national areas of countries in the European Union, including new countries, were constructed. Data collection from the new countries was more straightforward due to the requirement for them to adhere to the nomenclature of territorial units for statistics (NUTS) levels. The lack of adherence to NUTS levels in the 'old' countries of the European Union continues to create problems. There remains an urgent need to introduce comprehensive subnational data collection on important public health topics such as obesity and smoking. [ABSTRACT FROM AUTHOR]