<jats:title>ABSTRACT</jats:title> <jats:p> QscR, a LysR-type regulator, is the major regulator of assimilatory C <jats:sub>1</jats:sub> metabolism in <jats:italic>Methylobacterium extorquens</jats:italic> AM1. It has been shown to interact with the promoters of the two operons that encode the majority of the serine cycle enzymes ( <jats:italic>sga-hpr-mtdA-fch</jats:italic> for the <jats:italic>qsc</jats:italic> 1 operon and <jats:italic>mtkA-mtkB-ppc-mclA</jats:italic> for the <jats:italic>qsc</jats:italic> 2 operon), as well as with the promoter of <jats:italic>glyA</jats:italic> and its own promoter. To obtain further insights into the mechanisms of this regulation, we mapped transcriptional start sites for the <jats:italic>qsc1</jats:italic> and <jats:italic>qsc2</jats:italic> operons and for <jats:italic>glyA</jats:italic> via primer extension analysis. We also identified the specific binding sites for QscR upstream of the <jats:italic>qsc1</jats:italic> and <jats:italic>qsc2</jats:italic> operons and <jats:italic>glyA</jats:italic> by DNase I footprinting. The QscR protected areas were located at nucleotides −216 to −165, nucleotides −59 to −26, and nucleotides −72 to −39 within the promoter-regulatory regions upstream of transcriptional starts of, respectively, <jats:italic>qsc1</jats:italic> , <jats:italic>qsc2</jats:italic> and <jats:italic>glyA</jats:italic> . To examine the nature of the metabolic signal that may influence QscR-mediated regulation of the serine cycle genes, <jats:italic>Pqsc1</jats:italic> :: <jats:italic>xylE</jats:italic> translational fusions were constructed and expression of XylE monitored in the wild-type strain, as well as in knockout mutants defective in a variety of methylotrophy functions. The data from these experiments pointed toward formyl-H <jats:sub>4</jats:sub> F being a coinducer of QscR and possibly the major signal in the regulation of the serine cycle in <jats:italic>M. extorquens</jats:italic> AM1. The ability of formyl-H <jats:sub>4</jats:sub> F to enhance the binding of QscR to a specific region upstream of one of the serine cycle operons was demonstrated in gel retardation experiments. </jats:p>