Introduction: International recommendations state that reference values for lung function should derive from cross-sectional studies of healthy nonsmokers and be renewed from time to time because of cohort effect and newer, more accurate, technical equipment. In 1986, the Danish Lung Society published reference values for spirometry based on 570 individuals aged 30-70 years. Objectives: To produce new reference values for lung function and to extend the existing values by including individuals between 20 and 30 years of age and older than 70 years of age. Methods: Two similar but independent studies was used: The 2001-2003 examination of the Copenhagen City Heart Study and the 2003-2010 examination of the Copenhagen General Population Study. Of a total of 69822 individuals, we included 11288 healthy never-smoking white individuals to produce the reference values: 6307 women and 4981 men, 20 years of age or older with adequate lung function. Results: We used sex-stratified multiple linear regression analysis to find prediction formulas for forced expiratory volume in 1s (FEV1), forced vital capacity (FVC) and FEV1/FVC adjusted for age and height. The cutoff value of normal lung function was defined as the fifth percentile (also named the 5% quantile) according to gender, age and height. The robustness of the data was tested and validated in several ways. Conclusion: Compared with the 1986 data, our 2001-2010 material contributes with a substantial number of individuals in the more extreme groups of age and height, and in general, our dataset shows that in most subgroups, the lung function level has improved during the last two decades. Please cite this paper as: Løkke A, Marott JL, Mortensen J, Nordestgaard BG, Dahl M and Lange P. New Danish reference values for spirometry. Clin Respir J 2013; 7: 153-167.