The pathway through which retinol (vitamin A) is converted to its active metabolite, all-trans-retinoic acid (atRA), and subsequent receptor-mediated regulation of gene transcription by atRA is essential for all mammal life stages. This pathway is required for normal embryonic development and maintenance of cellular phenotype in adult organisms; chemicals that cause even minor interference with its normal function are potential developmental and adult toxicants. A short-term (24 h) in vitro mode-of-action screen for detecting chemicals that disrupt this essential pathway is described. It uses the mouse pluripotent P19 stem cell in a 96-well format, RT-qPCR gene-expression assay that does not require RNA purification to detect chemicals that interfere with retinol-induced Hoxa1 gene expression, a target of retinol signaling in mammals. A total of 21 chemicals were screened at a single 45 μM concentration. Four chemicals known to disrupt the pathway in the rodent embryo (citral, disulfiram, and two rodent teratogens, nitrofen and bisdiamine) all significantly inhibited Hoxa1 upregulation by retinol. An additional four of seven chemicals with varying degrees of structural similarity to known disruptors or to the retinoid side chain, but not previously known to disrupt the pathway, were positive in the screen. The xenoestrogens, diethylstilbestrol, bisphenol A, 4-n-nonylphenol, and genistein and the phthalate esters, dibutyl phthalate and dipentyl phthalate, but not diethylhexyl phthalate, also significantly disrupted the pathway. Of the 21 chemicals tested, diethylstilbestrol was the only chemical that showed evidence in the MTT assay that cytotoxicity may have contributed to disruption of the pathway. Birth Defects Res (Part B) 98:276–282, 2013. © 2013 Wiley Periodicals, Inc.