Our objective was to investigate whether genetic variants of the uncoupling protein 1 (UCP1) gene are associated with juvenile-onset obesity or alterations in weight gain and insulin sensitivity in young healthy Caucasians. Single-strand conformation polymorphism and heteroduplex analysis of the coding region of the UCP1 gene was performed in 56 subjects randomly selected at the draft board examination from a cohort of 156 males with juvenile-onset obesity. Association studies of amino acid variants were undertaken in the cohort of males with juvenile-onset obesity, a cohort of 205 randomly selected control males, and a subgroup of this cohort comprising 76 lean subjects. Genetic variants of the coding region as well as a previously described a-->g nucleotide polymorphism of the 5'-flanking region of the UCP1 gene were examined for associations with accelerated weight gain or reduced sensitivity to insulin in a cohort of 380 young healthy Caucasians. The mutational analysis revealed five nucleotide substitutions that changed the sequence of UCP1, Arg/Trp40, Ala/Thr64, Val/Met137, Met/Leu229, and Lys/Asn257 and two nucleotide substitutions in the nontranslated region of exon 1. Among subjects with juvenile-onset obesity, the allelic frequencies of Ala/Thr64 and Met/Leu229 were both 8.2% (95% confidence interval: 5.1-11.3%) vs. 8.8% (6.0-11.6%) and 8.1% (5.3-10.9%), respectively, in the cohort of randomly selected control subjects. Among lean control subjects, the allelic frequencies of the polymorphisms were 8.2% (3.7-12.7%) and 5.6% (1.9-9.3%), respectively. In the cohort of young healthy subjects, measurements of obesity and insulin sensitivity did not differ between carriers of the Ala/Thr64 and Met/Leu229 variants and wild-type carriers. The Val/Met137 and Lys/Asn257 mutations were each found in one subject with juvenile-onset obesity, and the Arg/Trp40 mutation was found in two obese subjects and in one control subject. The allelic frequency of the nucleotide polymorphism of the 5'-flanking region of the UCP1 gene was 25.3% (22.2-28.4%) in the cohort of 380 young Danes. There were no differences in body mass index, fat mass, waist-to-hip ratio, or weight gain during childhood or adolescence between carriers and noncarriers of this nucleotide variant. Although we cannot exclude an effect of the rare mutations in the UCP1 gene on susceptibility to juvenile-onset obesity, genetic variation of the coding region of the UCP1 gene is not a common factor contributing to obesity in Caucasian subjects of Danish ancestry.