Identifying the sequences that direct the spatial and temporal expression of genes and defining their function in vivo remains a significant challenge in the annotation of vertebrate genomes. One major obstacle is the lack of experimentally validated training sets. In this study, we made use of extreme evolutionary sequence conservation as a filter to identify putative gene regulatory elements, and characterized the in vivo enhancer activity of a large group of non-coding elements in the human genome that are conserved in human-pufferfish, Takifugu (Fugu) rubripes, or ultraconserved in human-mouse-rat. We tested 167 of these extremely conserved sequences in a transgenic mouse enhancer assay. Here we report that 45% of these sequences functioned reproducibly as tissue-specific enhancers of gene expression at embryonic day 11.5. While directing expression in a broad range of anatomical structures in the embryo, the majority of the 75 enhancers directed expression to various regions of the developing nervous system. We identified sequence signatures enriched in a subset of these elements that targeted forebrain expression, and used these features to rank all approximately 3,100 non-coding elements in the human genome that are conserved between human and Fugu. The testing of the top predictions in transgenic mice resulted in a threefold enrichment for sequences with forebrain enhancer activity. These data dramatically expand the catalogue of human gene enhancers that have been characterized in vivo, and illustrate the utility of such training sets for a variety of biological applications, including decoding the regulatory vocabulary of the human genome.