We evaluated the accuracy of a submaximal cycle ergometer test to assess cardiorespiratory fitness in adolescents and provided population-based reference values. In a health study of the Northern Finland birth cohort born in 1985 - 1986 (NFBC 1986), cardiorespiratory fitness of 5375 males and females aged 15 to 16 years was measured by a submaximal cycle ergometer test by using a two-stage exercise protocol designed for this survey. A total of 4903 subjects performed two work stages. Maximal workload and peak oxygen consumption were calculated on the basis of heart rate responses by the WHO extrapolation method. An additional 472 subjects were able to finish only one work stage. For them, peak oxygen uptake was calculated by the Astrand nomogram. To evaluate the accuracy of submaximal testing, a validation sample of 90 subjects carried out both a submaximal test and a maximal cycle ergometer test with direct measurement of peak oxygen uptake. The WHO method proved to estimate peak oxygen uptake with reasonable accuracy. The Astrand nomogram method overestimated peak oxygen uptake considerably, and therefore a new single-stage regression method was applied to calculate maximal workload and peak oxygen consumption. Reference values of cardiorespiratory fitness were provided for 2690 males and 2685 females aged 15 to 16 years.