Abstract: Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) and rhamnolipids produced by Pseudomonas aeruginosa share 3-hydroxyalkanoates (3HAs) as common structural units. Previously, we demonstrated their simultaneous production to share the cultivation cost for the intracellular products, PHAs, with the extracellular products, rhamnolipids. Here, we investigated the effects of temperature and carbon length of fatty acid substrates on the simultaneous production by P. aeruginosa IFO3924. The optimum temperatures for PHA and rhamnolipid syntheses were 30°C and 28°C, respectively, suggesting that the product ratio between these two products can be controlled by changing temperature. After the induction periods, both PHAs and rhamnolipids were produced at constant rates by resting cells. The apparent activation energies were determined as 45kJ/mol for PHA synthesis and 40kJ/mol for rhamnolipid synthesis from Arrhenius plots. These values in a similar level suggest that a common reaction such as β-oxidation before the synthesis of 3HA units is the rate-determining step. PHAs and rhamnolipids synthesized from fatty acids of even-number carbon length (Ceven) consisted of only Ceven-3HAs. By contrast, PHAs and rhamnolipids synthesized from fatty acids of odd-number carbon length (Codd) consisted of not only Codd-3HAs but also Ceven-3HAs. This is the first report to show the incorporation of Codd-3HAs into rhamnolipids. [ABSTRACT FROM AUTHOR]