Retinoids have been implicated in pattern formation processes in both developing chick limbs and in the regenerating limbs of urodele amphibians as well as in other aspects of embryonic development. Since chick wing buds have been shown to have a higher concentration of all-trans-retinoic acid (RA) in the posterior region than in the anterior region, we set out to look for a gradient of RA in the regenerating limb of the axolotl, Ambystoma mexicanum. We used high-performance liquid chromatography to separate, identify, and measure the concentration of retinoids present in the tissues. Our results show that the concentration of RA is about five times higher in posterior quarters of the limb regeneration blastema compared to anterior quarters. In addition, levels of RA are about two and a half times higher in blastemas from the radius-ulna level of the limb compared to those from the humerus level. By contrast, the limb regenerates of the African clawed frog, Xenopus laevis (which produce a regenerative outgrowth which is pattern-deficient), do not exhibit an anteroposterior gradient of any of the retinoids investigated. This paper also reports on the levels of retinoids in a number of other tissues as well. This anteroposterior gradient of RA in the axolotl limb regeneration blastema has significant implications for pattern formation during limb development and regeneration.