The internal organs of all vertebrates are asymmetrically organised across the left–right axis. The development of this asymmetry is controlled by a molecular pathway that includes the signalling molecule Nodal and the transcription factor Pitx2, proteins encoded by genes that are predominantly expressed on the left side of all vertebrate embryos studied to date. Vertebrates share Phylum Chordata with two other groups of animals, amphioxus and the urochordates (including ascidians). Both these taxa develop left–right asymmetries, and recent studies have begun to address the degree of conservation of nodal and Pitx2 in this process. Pitx2 is a member of the Pitx homeobox gene family, and in both amphioxus and ascidians Pitx gene expression is predominantly left sided. These studies suggest that left–right asymmetry in all chordates is regulated by a conserved developmental pathway, and that this pathway evolved before the separation of the lineages leading to living chordates. BioEssays 24:1004–1011, 2002. © 2002 Wiley-Periodicals, Inc.