Many algorithms analyze enhancers for overrepresentation of known and novel motifs, with the goal of identifying binding sites for direct regulators of gene expression. Twine is a Java GUI with multiple graphical representations (Views) of enhancer alignments that displays motifs, as IUPAC consensus sequences or position frequency matrices, in the context of phylogenetic conservation to facilitate cis-regulatory element discovery. Thresholds of phylogenetic conservation and motif stringency can be altered dynamically to facilitate detailed analysis of enhancer architecture. Views can be exported to vector graphics programs to generate high-quality figures for publication. Twine can be extended via Java plugins to manipulate alignments and analyze sequences.