Atomic force microscopy (AFM) was used to study the formation and growth of poly[(R)-3-hydroxybutyrate] (PHB) structures formed in the enzymatic polymerization of (R)-3-hydroxybutyryl coenzyme A [(R)-3-HBCoA] in vitro. Poly(3-hydroxyalkanoate) (PHA) synthase (PhaC(Re)) from Ralstonia eutropha, a class I synthase, was purified by one-step purification and then used for in vitro reactions. Before the reaction, PhaC(Re) molecules were deposited on highly oriented pyrolytic graphite (HOPG) and observed as spherical particles with an average height of 2.7 +/- 0.6 nm and apparent width of 24 +/- 3 nm. AFM analysis during the initial stage of the reaction, that is, after a small amount of (R)-3-HBCoA had been consumed, showed that the enzyme molecules polymerize (R)-3-HBCoA and form flexible 3HB polymer chains that extend from the enzyme particles, resulting in the formation of an enzyme-nascent PHB conjugate. When a sufficient amount of (R)-3-HBCoA was used as substrate, the reaction rapidly increased after the first minute followed by a slow increase in rate, and substrate was completely consumed after 4 min. After 4 min, spherical granules continued to grow in size to form clusters over 10 um in width, and in later stages of cluster formation, the cluster developed small projections with a size of approximately 100-250 nm, suggesting qualitative changes of the PHB clusters. Moreover, the high-resolution AFM images suggested that globular structures of approximately 20-30 nm apparent width, which corresponds to the size of PhaC(Re), were located on the surface of the small PHB granule particles.