As a first step in characterizing the genes involved in O-side chain synthesis in <i>Brucella abortus</i> strain 2308, a portion of the genomic DNA was cloned from a rough mutant created by Tn5 (KnR) mutagenesis. This mutant was rough based on the lack of reactivity by either whole cells or extracted LPS to an O-side chain monoclonal antibody (BRU-38). A 30 kb <i>Xba</i>I genomic fragment (including Tn5) from the rough strain was subcloned into a sequencing vector to create pJM6. When <i>B. abortus</i> 2308 was electroporated with pJM6, KnR clones were unable to react with BRU-38; a Southern analysis of these clones revealed Tn5 in the 30 kb <i>Xba</i>I genomic fragment. Various regions of the 30kb fragment were subcloned and tested for their ability to complement specific <i>rfa</i> and <i>rfb</i> mutants of <i>Escherichia coli</i> and <i>Salmonella typhimurium</i>. One particular DNA fragment complemented an <i>rfbD</i> mutation in <i>E. coli</i> as judged by agglutination with <i>E. coli</i> anti-O (0:85) serum. The same DNA fragment failed to cause <i>E. coli rfbD</i> to react with either BRU-38 or <i>B. abortus</i> anti-O polyclonal antisera. The <i>B. abortus</i> 30 kb <i>Xba</i>I fragment contains a gene which has been identified by comple-mentation as containing the equivalent of the <i>rfbD</i> gene encoding dTDP-rhamnose synthetase in <i>E. coli</i>. Since <i>Brucella</i> is not known to have rhamnose in its core this enzyme may have a different function in <i>Brucella</i> LPS synthesis. / Master of Science