Trisomy 21 is the prototype of human aneuploidies. Since its discovery in 1959, the hypothesis has been that overexpression of the approximately 230 human chromosome 21 (Hsa21) genes result in the complex phenotype. However, the level of overexpression of Hsa21 genes in trisomic individuals is presently unknown. We have used Taqman real-time quantitative PCR to accurately measure expression of the mouse orthologs of Hsa21 in the partial trisomy mouse model Ts65Dn. The transcript levels of 78 protein-coding genes present in three copies in Ts65Dn and 21 control genes were compared between Ts65Dn and normal mouse littermates. The mean overexpression of the aneuploid genes is very close to the expected 1.5-fold in all six tissues studied. However, only approximately a third of the genes (37%) are expressed at the theoretical value of 1.5-fold. On average, 45% of the genes are expressed at significantly lower than 1.5-fold, and 9% are not significantly different from 1.0. Interestingly, 18% of the aneuploid genes were expressed at levels significantly greater than 1.5-fold. These data provide candidate genes that might be involved in the phenotypes of Down syndrome, and reveal a complex regulation of gene expression that is not only related to gene copy number.