Conserved noncoding elements (CNEs) constitute the majority of sequences under 
purifying selection in the human genome, yet their function remains largely 
unknown. Experimental evidence suggests that many of these elements play 
regulatory roles, but little is known about regulatory motifs contained within 
them. Here we describe a systematic approach to discover and characterize 
regulatory motifs within mammalian CNEs by searching for long motifs (12-22 nt) 
with significant enrichment in CNEs and studying their biochemical and genomic 
properties. Our analysis identifies 233 long motifs (LMs), matching a total of 
approximately 60,000 conserved instances across the human genome. These motifs 
include 16 previously known regulatory elements, such as the histone 3'-UTR 
motif and the neuron-restrictive silencer element, as well as striking examples 
of novel functional elements. The most highly enriched motif (LM1) corresponds 
to the X-box motif known from yeast and nematode. We show that it is bound by 
the RFX1 protein and identify thousands of conserved motif instances, suggesting 
a broad role for the RFX family in gene regulation. A second group of motifs 
(LM2*) does not match any previously known motif. We demonstrate by biochemical 
and computational methods that it defines a binding site for the CTCF protein, 
which is involved in insulator function to limit the spread of gene activation. 
We identify nearly 15,000 conserved sites that likely serve as insulators, and 
we show that nearby genes separated by predicted CTCF sites show markedly 
reduced correlation in gene expression. These sites may thus partition the human 
genome into domains of expression.