Washed bottom-fraction particles (WBPs), that is, intact membrane-bound vesicles, prepared from the bottom fraction of centrifuged fresh latex, were shown to be active in in vitro rubber biosynthesis (RB) assays. The RB activity catalyzed by WBP enzymes was confirmed by enzyme parameter criteria. A washed bottom-fraction particle membrane (WBM), prepared from WBPs, was even more active in in vitro RB activity. Mg2+ was a cofactor required for RB enzymes, and the cation chelators EDTA and EGTA inhibited the RB process. The temperature had a strong effect on RB stimulation during the heat pretreatment of the WBM before RB assays. A detergent also proved to a strong RB stimulator. The anionic detergent SDS, above the critical micelle concentration, was a strong stimulator of WBM activity. This was not observed with the nonionic detergents Triton X-100 and Tween 20. Various temperature ranges used for WBM preincubation showed a sharp rise in RB activity above 70degreesC. The temperature required for a sharp rise in RB was lowered substantially in the presence of SDS. It was down to 40degreesC with the combined preincubation but remained constant with an even higher RB activity. Greater rubber formation at a lower temperature was observed in the presence of SDS. A synergistic effect for RB stimulation appeared during the heat pretreatment of WBM in the presence of SDS.