Transmission electron microscopy (TEM) and Cryo-TEM were used to study the growth kinetics of poly(3-hydroxybutyrate) granules produced by in vitro polymerization. The in vitro formation of poly(3-hydroxybutyrate) uses a recombinant form of the PHA synthase to polymerize [R]-(-)-3-hydroxybutyryl-CoA. Since the in vitro reaction contains only synthase and monomer, it is a simpler system than the in vivo biosynthesis of poly(3-hydroxybutyrate). TEM and Cryo-TEM were used in conjunction with image analysis to examine the granules that were formed in the in vitro reaction. The in vitro reaction yielded spherical granules of rapidly increasing size; the initially observed granules were already larger than 0.1 mu m. While the average granule diameter and volume increased with reaction time, the number of granules decreased throughout the reaction due to coalescence. Basic kinetic parameters, including K-M and V-max were determined and compared to those reported for the in vivo biosynthesis of poly(3-hydroxybutyrate). In addition it was found that the granules formed by this process were essentially noncrystalline. A computer simulation of the reaction, based on initial formation of relatively large microporous granules that consolidate by expulsion of water during polymerization, accounted for the shape of the kinetic curves.