Transcription factors regulate gene expression in development by binding DNA at recognised motifs. Regulation can be diversified by using divergent, low-affinity motifs, but some transcription factors may also recognise multiple distinct motifs. Now, Izumi Oda-Ishii, Deli Yu, and Yutaka Satou report that the ascidian Zic transcription factor Zic-r.a specifies posterior fate in early embryos and activates neural genes in late embryos via two, cell lineage-specific recognition motifs. The authors first show that expression of the direct Zic-r.a target Tbx6-r.b is regulated differently in two muscle lineages, B5.1 and B6.4 (in B6.4, its expression is independent of β-catenin/Tcf7). Computational and in vitro analyses reveal the presence of a second, weaker affinity non-canonical Zic-r.a-binding motif in the 189 bp upstream sequence of Tbx6-r.b. The higher concentration of Zic-r.a in the B6.4 lineage appears to explain the β-catenin-independent expression of Tbx6-r.b. In a distinct lineage, zygotically expressed Zic.r.a regulates neural genes via the canonical motif. Finally, the authors show that distinct zinc-finger domains are responsible for the recognition of canonical and non-canonical motifs in target genes. A key developmental transcription factor in ascidian embryos therefore binds two distinct recognition motifs at different affinities and in different ways.