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50 million years ago, has not been reversed despite subsequent diversification of feeding modes and retention of pharyngeal teeth. We asked whether the cis-regulatory region of a gene whose expression loss parallels cypriniform tooth loss, Dlx2b, retains the capacity for expression in oral teeth. We first created a zebrafish reporter transgenic line that recapitulates endogenous dlx2b expression. We then showed that this zebrafish construct drives reporter expression in oral teeth of the related characiform Astyanax mexicanus. This result, along with our finding that Dlx genes are required for normal tooth development, suggests that changes in trans-acting regulators of these genes were responsible for loss of cypriniform oral teeth. Preservation of oral enhancer function unused for >50 million years could be the result of pleiotropic function in the pharyngeal dentition. If enhancers of other genes in the tooth developmental pathway are similarly preserved, teeth lost from specific regions may be relatively easy to reacquire in evolution. © 2006 by The National Academy of Sciences of the USA.">