We have previously shown differential regulation of components of the Retinoic acid (RA) pathway in Xenopus tadpole hindlimb regeneration. RA is thought to act as a morphogen, providing positional information during development and regeneration. We have investigated the regulation of genes involved in RA synthesis, catabolism, and binding in developing and regenerating Xenopus limbs. Our data indicate that RA is synthesised by Raldh2 in proximal cells during limb bud outgrowth. Furthermore, Cyp26b is expressed transiently in the progress zone of developing limbs and the blastema of regenerating limbs suggesting degradation of RA occurs in both processes. The RA-binding protein Crabp2 is also upregulated during regeneration. We summarise this data to predict the presence of evolving gradients of RA in the developing amphibian limb. Thus, RA from the stump cells could be responsible for the establishment of proximal-distal pattern during limb regeneration, as predicted by classical studies. Developmental Dynamics 240:1259–1270, 2011. © 2011 Wiley-Liss, Inc.