A newly developed in vitro activity assay for medium-chain-length(mcl)-poly(3-hydroxyalkanoate) polymerases is described. Polymerase C1 of Pseudomonas oleovorans GPo1 attached to isolated granules was used as model enzyme. A direct correlation was found between (R)-3-hydroxyoctanoylCoA depletion and poly(3-hydroxyalkanoate) synthesis due to polymerase C1 activity. Highest activities of 1.13 U/mg granule bound protein and highest specific activities of 2.3 U/mg polymerase C1 were determined towards (RS)-3-hydroxyoctanoylCoA. A first determination of a Km value for mcl poly(3-hydroxyalkanoate) polymerases was performed leading to an estimated Km of 0.16 (± 0.1) mM for granule bound polymerase C1 with (RS)-3-hydroxyoctanoylCoA as substrate. Polymerase C1 showed no activity towards (RS)-3-hydroxybutyrylCoA and a specific activity of 0.28 U/mg polymerase C1 for (R)-3-hydroxyvalerylCoA. (R)-3-HydroxyoctanoylCoA and a mixture of (RS)-3-hydroxyoctanoylCoA were both depleted for more than 75% by granule-bound polymerase C1, suggesting a non-rate-limiting epimerase activity attached to poly(3-hydroxyalkanoate) granules isolated from Pseudomonas putida GPp104[pGEc405]. Whereas no relationship was found between the activity of granule-bound polymerase C1 and poly(3-hydroxyalkanoate) content of the granules, higher activities were measured when a higher substrate concentration or more enzyme was present in the in vitro activity assay.