The aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of the Per‐ARNT‐Sim (PAS) protein superfamily, transcription factors that mediate the cellular responses to various developmental signals and environmental conditions. A β‐class (“partner”) PAS protein, ARNT exhibits the capacity to form transcriptionally active heterodimers with several α‐class (“sensor”) proteins, including the aryl hydrocarbon receptors (AHRs), the hypoxia‐inducible factors (HIFs), and the Single minded (Sim) proteins. Two genes encode different forms of ARNT in mammals: ARNT1, which is widely expressed, and ARNT2, which is limited to the brain and kidneys of adults and specific neural and branchial tissues of embryos. In contrast, fish apparently express only a single ARNT gene, although in different species, this may be either ARNT1 or ARNT2. In efforts to understand the evolution of ARNT proteins throughout the vertebrate lineage, we isolated an ARNT cDNA from early life stages of the amphibian Xenopus laevis. The encoded protein binds cognate DNA sequences in concert with mouse AHR. Phylogenetic analysis reveals that this sequence is orthologous to mammalian ARNT2 and paralogous to the recently reported X. laevis ARNT1. ARNT2 mRNA expression begins later than ARNT1 (stage 22 vs. stage 8), suggesting the two proteins play distinct roles during development. Hence, in the expression of two well‐conserved ARNT paralogs with distinct expression patterns, X. laevis resembles mammals rather than fish. Diversity in the number and function of PAS proteins, including ARNT, may underlie significant species differences in developmental programming and biochemical response to environmental conditions. The identification of multiple amphibian ARNT paralogs represents an important step in the understanding of evolution and functional variation of ARNT in vertebrates. J. Exp. Zool. (Mol. Dev. Evol.) 300B:48–57, 2003. © 2003 Wiley‐Liss, Inc.