MicroRNAs (miRNAs) are conserved non-coding small RNAs with potent post-transcriptional gene regulatory functions. Recent computational approaches and sequencing of small RNAs had indicated the existence of about 80 Ciona intestinalis miRNAs, although it was not clear whether other miRNA genes were present in the genome. We undertook an alternative computational approach to look for Ciona miRNAs. Conserved non-coding sequences from the C. intestinalis genome were extracted and computationally folded to identify putative hairpin-like structures. After applying additional criteria, we obtained 458 miRNA candidates whose sequences were used to design a custom microarray. Over 100 of our predicted hairpins were identified in this array when probed with RNA from various Ciona stages. We also compared our predictions to recently deposited sequences of Ciona small RNAs and report that 170 of our predicted hairpins are represented in this data set. Altogether, about 250 of our 458 predicted miRNAs were represented in either our array data or the small-RNA sequence database. These results suggest that Ciona has a large number of genomically encoded miRNAs that play an important role in modulating gene activity in developing embryos and adults.