Somitogenesis is the key developmental process that lays down the framework for a metameric body in vertebrates. Somites are generated from the un-segmented presomitic mesoderm (PSM) by a pre-patterning process driven by a molecular oscillator termed the segmentation clock. The Delta-Notch intercellular signaling pathway and genes belonging to the hairy (h) and Enhancer of split (E(spl))-related (h/E(spl)) family of transcriptional repressors are conserved components of this oscillator. A subset of these genes, called cyclic genes, is characterized by oscillating mRNA expression that sweeps anteriorly like a wave through the embryonic PSM. Periodic transcriptional repression by H/E(spl) proteins is thought to provide a critical part of a negative feedback loop in the oscillatory process, but it is an open question how many cyclic h/E(spl) genes are involved in the somitogenesis clock in any species, and what distinct roles they might play. From a genome-wide search for h/E(spl) genes in the zebrafish, we previously estimated a total of five cyclic members. Here we report that one of these, the mHes5 homologue her15 actually exists as a very recently duplicated gene pair. We investigate the expression of this gene pair and analyse its regulation and activity in comparison to the paralogous her12 gene, and the other cyclic h/E(spl) genes in the zebrafish. The her15 gene pair and her12 display novel and distinct expression features, including a caudally restricted oscillatory domain and dynamic stripes of expression in the rostral PSM that occur at the future segmental borders. her15 expression stripes demarcate a unique two-segment interval in the rostral PSM. Mutant, morpholino, and inhibitor studies show that her12 and her15 expression in the PSM is regulated by Delta-Notch signaling in a complex manner, and is dependent on her7, but not her1 function. Morpholino-mediated her12 knockdown disrupts cyclic gene expression, indicating that it is a non-redundant core component of the segmentation clock. Over-expression of her12, her15 or her7 disrupts cyclic gene expression and somite border formation, and structure function analysis of Her7 indicates that DNA binding, but not Groucho-recruitment seems to be important in this process. Thus, the zebrafish has five functional cyclic h/E(spl) genes, which are expressed in a distinct spatial configuration. We propose that this creates a segmentation oscillator that varies in biochemical composition depending on position in the PSM. © 2007 Elsevier Inc. All rights reserved.