The mechanism involved in the crystallization of bacterial polyhydroxybutyrate (PHB) and poly(hydroxybutyrate-co-hydroxyhexanoate) P(HB-co-HH) induced by orotic acid as a nucleant was investigated by using differential scanning calorimetry (DSC), gel permeation chromatography (GPC) and proton nuclear magnetic resonance spectroscopy (H-1-NMR). GPC measurements both carried on solvent cast and hot pressed samples did not show any significant drop of the molecular weight caused by the addition of the nucleant, indicating that no chemical reaction happened during the nucleation process. This result was confirmed by H-1-NMR analysis of oligohydroxybutyrate (OHB) treated with an excess amount of orotic acid. The possibility of epitaxial growth of the polymer crystal on the surface of the nucleant crystal was then investigated. It was found that there is an outstanding crystalline lattice matching between the plane (100) of the PHB crystal and the plane (007) of the orotic acid crystal. In comparison, the matching obtained with conventional nucleating agents, such as boron nitride and talc, was worse. Moreover, some regular hydrogen bonds between the polyester and orotic acid could stabilize the physical process. According to these results, the physical mechanism involving the epitaxial matching between orotic acid and PHB appears to be the most probable nucleation mechanism. (C) 2009 Wiley Periodicals, Inc. J Appl Polym Sci 115: 709-715, 2010