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The objective of this study was to determine whether variation in energy expenditure created by selection on heat loss is mediated by uncoupling protein-1 (UCP1) in brown adipose tissue. Divergent selection for heat loss developed lines of mice with high (MH) and low (ML) maintenance energy expenditure. Concentration of UCP1 mRNA in brown adipose tissue (BAT) was 93% greater in ML than in MH mice (P < 0.02). Two new lines of mice, KH and KL, were bred by backcrossing a UCP1 knockout gene into the MH and ML lines, respectively; KH and KL with both knockout (−/−) and wild type (+/+) UCP1 genotypes were generated. At 13 wk of age, KH mice exhibited greater heat loss (166 kcal∙kg0.75∙d−1) than KL mice (126.4 kcal∙ kg0.75∙d−1) regardless of the UCP1 knockout (P < 0.0001). Concentration of UCP2 mRNA in BAT was 74% greater in UCP1 knockout mice (−/−) than in wild type (+/+; P = 0.0001). We conclude that response to selection for increased energy expenditure was not mediated by increased expression or function of UCP1.