The polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC(Re)) shows a lag time for the start of its polymerization reaction, which complicates kinetic analysis of PhaC(Re). In this study, we found that the lag can be virtually eliminated by addition of 50 mg/L TritonX-100 detergent into the reaction mixture, as well as addition of 2.5 g/L Hecameg detergent as previously reported by Gerngross and Martin (Proc Natl Sci USA 92: 6279-6283, 1995). TritonX-100 is an effective lag eliminator working at much lower concentration than Hecameg. Kinetic analysis of PhaC(Re) was conducted in the presence of TritonX-100, and PhaC(Re) obeyed Michaelis-Menten kinetics for (R)-3-hydroxybutyryl-CoA substrate. In inhibitory assays using various compounds such as adenosine derivatives and CoA derivatives, CoA free acid showed competitive inhibition but other compounds including 3'-dephospho CoA had no inhibitory effect. Furthermore, PhaC(Re) showed a considerably reduced reaction rate for 3'-dephospho (R)-3-hydroxybutyryl CoA substrate and did not follow typical Michaelis-Menten kinetics. These results suggest that the 3'-phosphate group of CoA plays a critical role in substrate recognition by PhaC(Re).