Objective To compare the Danish Physical Activity Questionnaire (DPAQ) estimating physical activity energy expenditure (PAEE) and physical activity level (PAL) and the pattern of physical activity (including health-related physical activity) with measurements from a validated position and motion instrument (ActiReg((R))). Methods One hundred and thirty-eight healthy Danish volunteers aged 20-59 years participated. Participants filled in the DPAQ at the end of the day every day for seven consecutive days and carried the ActiReg((R)) on the same days as the DPAQ were filled in. Results A small, but statistically significant difference was seen between the DPAQ and the ActiReg((R)) for PAEE (mean: -0.32 MJ center dot d(-1); 95% limits of agreement: ((-2.88)-2.24 MJ center dot d(-1)); P = 0.003) and PAL (-0.03; ((-0.37)-0.31); P = 0.02) for the whole group and for women (P <0.008 for PAEE and PAL), but not for men. The correlation between methods was good for PAEE (r = 0.71, P <0.001) and PAL (r = 0.64, P <0.001). No difference was observed for time spent in moderate plus vigorous physical activity (MVPA) (P = 0.40). Time reported in MVPA with the DPAQ was positively correlated with time spent in MVPA measured by the ActiReg((R)) (rho = 0.53, P <0.001). Conclusions Although the volunteer sample may influence the representativeness of the results, the DPAQ provided a close estimate of PAEE, PAL (2-5% underestimation) and health-related physical activity (MVPA) in healthy adults at group level. The results indicate that the questionnaire can be used to rank individuals according to energy expenditure and level of total physical activity and to provide information on health-related physical activity.