Natural rubber from Hevea brasiliensis is a high molecular mass polymer of isoprene units with cis-configuration. The enzyme responsible for the cis-1,4-polymerization of isoprene units has been idengified as a particle-bound rubber transferase, but no gene encoding this enzyme has been cloned from rubber-producing plants. By using sequence information from the conserved regions of cis-prenyl chain elongating enzymes that were cloned recently, we have isolated and characterized cDNAs from H. brasiliensis for a functional factor participating in natural rubber biosynthesis. Sequence analysis revealed that all of the five highly conserved regions among cis-prenyl chain elongating enzymes were found in the protein sequences of the Hevea cis-prenyltransferase. Northern blot analysis indicated that the transcript(s) of the Hevea cis-prenyltransferase were expressed predominantly in the latex as compared with other Hevea tissues examined. In vitro rubber transferase assays using the recombinant gene product overexpressed in Escherichia coli revealed that the enzyme catalyzed the formation of long chain polyprenyl products with approximate sizes of 2 × 103−1 × 104 Da. Moreover, in the presence of washed bottom fraction particles from latex, the rubber transferase activity producing rubber product of high molecular size was increased. These results suggest that the Hevea cis-prenyltransferase might require certain activation factors in the washed bottom fraction particles for the production of high molecular mass rubber.