Individuals with uniparental disomy of chromosome 14 (Temple and Kagami-Ogata syndromes) exhibit a number of developmental abnormalities originating, in part, from aberrant developmental expression of imprinted genes in the DLK1-DIO3 cluster. Although genomic imprinting has been reported in humans for some genes in the cluster, little evidence is available about the imprinting status of DIO3, which modulates developmental exposure to thyroid hormones. We used pyrosequencing to evaluate allelic expression of DLK1 and DIO3 in cDNAs prepared from neonatal foreskins carrying single-nucleotide polymorphisms (SNPs) in the exonic sequence of those genes, and hot-stop PCR to quantify DIO3 allelic expression in cDNA obtained from a skin specimen collected from an adult individual with known parental origin of the DIO3 SNP. In neonatal skin, DLK1 and DIO3 both exhibited a high degree of monoallelic expression from the paternal allele. In the adult skin sample, the allele preferentially expressed is that inherited from the mother, although a different, larger DIO3 mRNA transcript appears the most abundant at this stage. We conclude that DIO3 is an imprinted gene in humans, suggesting that alterations in thyroid hormone exposure during development may partly contribute to the phenotypes associated with uniparental disomy of chromosome 14.