In vivo random mutagenesis of the polyhydroxyalkanoate (PHA) synthase gene from Aeromonas punctata was performed employing the mutator strain Escherichia coli XL1-Red. About 200,000 mutants were screened on Nile red-containing medium and five mutants with enhanced fluorescence were selected. Four of these mutants exhibited enhanced in vivo and in vitro PHA synthase activity. Mutant M1, which carried the single mutation F518I, showed a five-fold increase in specific PHA synthase activity, whereas the corresponding mediated PHA accumulation increased by 20%, as compared with the wild-type PHA synthase. Mutant M2, which carried the single mutation V214G, showed a two-fold increase in specific PHA synthase activity and PHA accumulation only increased by 7%. Overall, the in vitro activities of the overproducing mutants ranged from 1.1- to 5-fold more than the wild-type activity, whereas the amounts of accumulated PHA ranged over 107-126% of that of the wild type. Moreover, all mutants mediated synthesis of PHAs with an increased weight average molar mass, but the molar fractions of 3-hydroxybutyrate and 3-hydroxyhexanoate remained almost constant. In vivo random mutagenesis proved to be a versatile tool to isolate mutants exerting improved properties with respect to PHA biosynthesis.