A novel and efficient method for the production of enantiomerically pure (R)-(-)-hydroxycarboxylic acids by in vivo depolymerization of microbial polyester polyhydroxyalkanoates (PHAs) was developed. Using this method, several model compounds, (R)-(-)-3-hydroxyalkanoic acids, consisting of 4 to 12 carbon atoms, and (R)-(-)-3-hydroxy-5-phenylvaleric acid, could be prepared. In particular, (R)-(-)-3-hydroxybutyric acid could be efficiently prepared by this method. By providing the environmental condition in which cells possess high activity of intracellular PHA depolymerase and low activity of (R)-(-)-3-hydroxybutyric acid dehydrogenase, (R)-(-)-3-hydroxybutyric acid could be produced with a yield of 96% in only 30 min by in vivo depolymerization of polyhydroxybutyrate (PHB) accumulated in Alcaligenes latus. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 363–368, 1999.