We show that sequence comparisons at different levels of resolution can efficiently guide functional analyses of regulatory regions in the ascidians Ciona savignyi and Ciona intestinalis. Sequence alignments of several tissue-specific genes guided discovery of minimal regulatory regions that are active in whole-embryo reporter assays. Using the Troponin I (TnI) locus as a case study, we show that more refined local sequence analyses can then be used to reveal functional substructure within a regulatory region. A high-resolution saturation mutagenesis in conjunction with comparative sequence analyses defined essential sequence elements within the TnI regulatory region. Finally, we found a significant, quantitative relationship between function and sequence divergence of noncoding functional elements. This work demonstrates the power of comparative sequence analysis between the two Ciona species for guiding gene regulatory experiments.