Previous studies have postulated that Xenopus embryos contain an endogenous retinoic acid (RA) concentration gradient from posterior to anterior during the process of primary axis formation, since RA is able to alter profoundly primary axis formation in Xenopus embryos, to increase the expression of some posterior markers, and to inhibit the expression of some anterior markers, including homeobox-containing genes. Here, we provide direct evidence for this hypothesis. By using a reporter cell system, we demonstrate that the endogenous biologically active retinoid concentration in whole Xenopus embryos increases 3-fold from the two-cell stage to the neurula stage, and that the active retinoid concentration in the dorsal marginal zone, a region wherein the Spemann's organizer is located, increases about 5-fold from the early gastrula to late gastrula stages, suggesting the developmental regulation of the retinoid levels. In the early neurula stage (stage 13-14), endogenous active retinoids are present in a concentration gradient with the highest level at the posterior end, about 10-fold higher than that at the anterior end, of the embryo. This concentration gradient may be established during gastrulation and may provide positional cues for primary axis formation.