dTDP-rhamnose is synthesized from dTTP and glucose-1-phosphate by four enzymatic steps in the gram-negative bacteria. By using a homologous PCR product, a gene cluster encoding four genes (rfbA, rfbB, rfbC, rfbD) involved in L-rhamnose biosynthesis by Acinetobacter calcoaceticus was isolated and sequenced. The four genes were clustered on the biosynthetic operon in the order of rfbB, D, A, C. A gene, rfbA, encoding glucose-l-phosphate thymidylyltransferase (RfbA), was cloned from A. calcoaceticus pathogenic and encapsulated in the gram-negative bacterium. This enzyme catalyzes the formation of dTDP-D-glucose From $alpha$-D-glucose-1-phosphate and dTTP.RfbA was amplified by PCR and inserted into the $T_7$ expression system. The activity of RfbA was determined by the capillary electrophoresis. The $K_m$ values for dTTP and $alpha$-D-glucose-1-phosphate were calculated to be 1.27 mM and 0.80 mM, respectively by using the Line-Weaver Burk plot. RfbA is inactivated by diethylpyrocarbonate.