Metabolic activation of retinol (vitamin A) via sequential actions of retinol and retinal dehydrogenases produces the active metabolite all-trans-retinoic acid. This work reports cDNA cloning, enzymatic characterization, function in a reconstituted path of all-trans-retinoic acid biosynthesis in cell culture, and mRNA expression patterns in adult tissues and embryos of a mouse retinol dehydrogenase, RDH1. RDH1 represents a new member of the short chain dehydrogenase/reductase superfamily that differs from other mouse RDH in relative activity with all-trans and cis-retinols. RDH1 has a multifunctional catalytic nature, as do other short chain dehydrogenase/reductases. In addition to retinol dehydrogenase activity, RDH1 has strong 3alpha-hydroxy and weak 17beta-hydroxy steroid dehydrogenase activities. RDH1 has widespread and intense mRNA expression in tissues of embryonic and adult mice. The mouse embryo expresses RDH1 as early as 7.0 days post-coitus, and expression is especially intense within the neural tube, gut, and neural crest at embryo day 10.5. Cells cotransfected with RDH1 and any one of three retinal dehydrogenase isozymes synthesize all-trans-retinoic acid from retinol, demonstrating that RDH1contributes to a path of all-trans-retinoic acid biosynthesis in intact cells. These characteristics are consistent with RDH1 functioning in a path of all-trans-retinoic acid biosynthesis starting early during embryogenesis.