The Human Genome Project has created a dire need for designing high throughput computational and functional tools that will link DNA sequences to their biological function. Whereas major efforts have been devoted to the identification of genes, much o f our genetic complexity is due to sophisticated regulatory networks that determine when and where genes are expressed. Regulatory elements, such as locus control regions (LCRs), that act over large genomic intervals to influence the expression patterns of genes have been difficult to identify using standard molecular biology approaches. I exploited a comparative sequence-based strategy to identify noncoding sequences with the physical properties of LCR elements in the human 5q31 interleukin gene cluster region. Comparative sequence analysis of 1 Mb of orthologous human (5q31) and mouse (chromosome 11) sequences identified 90 conserved noncoding elements (CNSs) (> 100 bp in length and > 70% identity). Evolutionary analysis of the IL-4 - IL-13 region with 200 kb of dog syntenic DNA established that all the CNS elements identified by human/mouse comparisons are also highly conserved in the dog. The largest noncoding sequence identified (401 bp in length) was conserved both with regard to sequence (~80% identity in mice, humans, cows, dogs, rabbits) and its genomic location (in the IL-4 — IL-13 intergenic region in the 4 mammals examined). Functional characterization of this element in human YAC transgenic mice revealed it to be a potent regulator of IL-4, IL-13 and IL-5 expression — genes spread over 120 kb. In addition to showing that comparative sequence analysis is a powerful tool that can be used to identify regulatory elements, and annotate noncoding DNA. I have also directed my efforts to designing a high throughput functional assay that will link evolutionarily conserved noncoding sequences to their biological function. Using this in vitro assay I functionally characterized all the conserved noncoding elements surrounding IL-4, IL-13 and IL-5, and established that three of them CNS-2, CNS-14 and CNS-17 are involved in T-cell specific regulation of these cytokines.