Pseudomonas putida GPo1 is able to accumulate polyhydroxyalkanoates (PHA) in the form of intracellular granules as storage materials. PHA granules were isolated and analyzed for protein activities. An acyl-CoA-synthetase (ACS1) activity was detected from the purified PHA granules. The corresponding gene acs1 was then cloned from P. putida GPo1. With the genomic walking technique, a homologue acs2 located upstream of acs1 was discovered and cloned. Fusions of both acs1 and acs2 with the gene encoding the green fluorescent protein (GFP) were constructed and expressed in GPo1. In vivo fluorescence microscopy studies showed that the fluorescence generated from the ACS1-GFP was mainly associated with the PHA granules, whereas that from ACS2-GFP was mainly with the membrane of the cells. In the control strain (containing GFP alone) fluorescence was distributed evenly in the cytoplasm. We concluded that ACS1 is located on the PHA granules and may play a central role in mobilization of PHA, for example, conversion of hydroxycarboxylic acid monomers to hydroxycarboxyl-CoA, which can be further utilized by the cells.