In three rubber-producing species, in vitro, the rates of initiation and polymerization and the biopolymer molecular weight produced were affected by the concentration of farnesyl diphosphate (FPP) initiator and isopentenyl diphosphate (IPP) elongation substrate (monomer). Ficus elastica, a low molecular weight-producer in vivo, synthesized rubber polymers approximately twice the molecular weight of those made by Hevea brasiliensis or Parthenium argentatum (which produce high molecular weights in vivo), possibly due to its lower IPP K-m. In all species, increasing FPP concentrations increased rubber biosynthetic rate and new molecules initiated but decreased molecular weight by competition with the allylic diphosphate (APP) end of elongating rubber molecules for the APP binding site. Increasing IPP concentrations increased rubber biosynthetic rate and rubber molecular weight, but only when FPP concentrations were below the FPP K-m's or where negative cooperativity operated. In conclusion, rubber transferase is not the prime regulator of rubber molecular weight in vivo.