a++Undecaprenyl pyrophosphate synthetase has been purified from Lactobacillus plantarum. It catalyzes the formation of a C55 polyprenyl pyrophosphate having isoprene residues with cis stereochemistry. The enzyme was shown to be an acidic protein (pI = 5.1), which can be partially purified by preparative gel electrophoresis and Blue-agarose column chromatography. The Km's of the enzyme for its substrates t,t-farnesyl pyrophosphate and isopentenyl pyrophosphate were determined to be 0.13 and 1.92 microM, respectively. The molecular weight of the enzyme was estimated by molecular sieve chromatography and gradient centrifugation to be 56,000 +/- 4000. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the protein was composed of a dimer of 30,000-Da subunits. The enzyme was inactivated by the arginine-specific reagents phenylglyoxal, butanedione and, cyclohexanedione, but this inactivation was not prevented by either of the substrates.