AbstractStudies using bioassays in normal mice and gene activation in transgenic reporter mice have demonstrated peaks of retinoic acid receptor (RAR) signaling in the brachial and lumbar regions of the spinal cord. Recently, Solomin et al. (Solomin et al. [] Nature 395:398–402) detected a retinoid X receptor (RXR) signal in the same region of the developing spinal cord at a slightly later stage than the RAR signal. This finding raises the question of which retinoid ligands underlie RAR and RXR signaling in this part of the embryo. Quantitative measurements of regional differences in retinoid profiles have not been reported previously due to limitation in the sensitivity and specificity of available retinoid detection methods. Here, by using a recently developed ultrasensitive HPLC technique (Sakhi et al. [] J. Chromatogr. A 828:451–460), we address this question in an attempt to identify definitively the endogenous retinoids present in different regions of the spinal cord at the stages when regional differences in RAR and RXR signaling have been reported. We find a bimodal distribution of all‐trans retinoic acid (at‐RA), the ligand for RARs, and relate this to the expression of several retinoid‐synthesizing enzymes. However, we do not detect 9‐cis‐retinoic acid (9‐cis‐RA), the putative RXR ligand, in any region of the spinal cord unless retinoid levels are massively increased experimentally by gavage feeding pregnant mice with teratogenic doses of at‐RA. This study provides for the first time quantitative profiles of endogenous retinoids along the axis of the developing spinal cord, thereby establishing a foundation for more definitive studies of retinoid function in the future. It sets definite limits on how much 9‐cis‐RA potentially is present and demonstrates that at‐RA predominates over 9‐cis‐RA by at least 30‐ to 180‐fold in different spinal cord regions. © 2001 Wiley‐Liss, Inc.