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The objectives of the study were to assess the 13C bicarbonate dilution technique using an automatic blood sampling system and to use this technique to estimate energy expenditure (EE) based on the CO2 production of 14 lactating Holstein cows on pasture or in a freestall barn. The effects of physical activity and eating behavior on EE were also assessed. Cows were exposed to each feeding system in a crossover design with two 14-d experimental periods, each consisting of an adaptation period and a 7-d data collection period. Cows either grazed on pasture or had ad libitum access, in the freestall barn, to grass cut daily from the same paddock. All cows were supplemented with a cereal-based concentrate. The EE of each cow was determined from 0700 to 1300 h on 1 d of each collection period. Blood samples for the 13C bicarbonate dilution technique were taken either manually in the barn or using an automatic blood sampling system on pasture. Eating pattern and physical activity were recorded from 0700 to 1300 h using a behavior recorder and an activity meter, respectively. Milk yield was recorded daily. Individual feed intake was estimated using the alkane double-indicator technique. Two preceding experiments confirmed that the sampling technique (manual or automatic) and the following storage of the blood samples (frozen directly after withdrawal or first cooled on ice and then frozen 6 h later) had no effect on 13CO2 enrichment in the extracted blood CO2 or on the subsequent calculation of CO2 production. During the 6-h measurement period, the EE of cows on pasture was higher than that of cows in the freestall barn. Daily feed intake and milk production were not affected by the feeding treatment. Grazing cows spent more time walking and less time standing and lying than did cows fed indoors. Time spent eating was greater and time spent ruminating was lower for cows on pasture compared with grass-fed cows in the barn. In conclusion, the 13C bicarbonate dilution technique, combined with an automatic blood sampling system, is a suitable method to determine the EE of lactating dairy cows on pasture. Positive correlations between EE and walking and eating time indicate that the higher energy requirements of dairy cows on pasture may be at least partly caused by a higher level of physical activity. However, before specific recommendations about additional energy supply can be given, it must be determined whether EE measured over 6 h can be extrapolated to 24 h. Furthermore, the apparent inconsistency between EE, feed intake, and milk production needs to be resolved., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)