AbstractParticular sequence motifs can act as transcription regulators. Because the total regulatory effects of such motifs can be related to their abundance, their presence might be expected at locations within the genome where sequences are repeated. Multiple repeats that vary in number among individuals occur within the ribosomal first internal transcribed spacer (ITS1) in some species in three trematode genera: Paragonimus, Schistosoma and Dolichosaccus. In all of these genera we found in ITS1, sequences identical to known enhancer motifs. We also searched for, and identified, known regulatory motifs in published ITS1 sequences of other parasitic flatworms including Echinostoma spp. (Trematoda) and Echinococcus spp. (Cestoda) which lack multiple repeats in ITS1. We present three lines of evidence that this widespread occurrence of such motifs within the ITS1 of parasitic flatworms may indicate a functional role in regulating tissue‐ or stage‐specific transcription of ribosomal genes. First, these motifs are identical to ones whose functional roles have been established using in vitro assays of transcriptional rates. Second, in all 18 species investigated here, between one and three different regulatory motifs were identified. In 14 of these 18 species, the probability that at least one of these motifs occurred because of the random assortment of bases within the regions investigated was 10% or less. In 12 of these 14 species, the probability was 5% or less. Third, the evolutionary divergence of flatworm species investigated is quite ancient. Therefore, the interspecific distribution of motifs observed here, in a rapidly evolving region such as ITS1, is unlikely to be attributable solely to shared evolutionary histories. These results, therefore, suggest a broader functional role for the ITS1 than previously thought. J. Exp. Zool. (Mol. Dev. Evol.) 296B:80–86, 2003. © 2003 Wiley‐Liss, Inc.