Pseudomonas oleovorans forms medium-chain-length poly(3-hydroxyalkanoate) (PHA) most effectively at growth rates below the maximum specific growth rate. Under adequate conditions, PHA accumulates rates in inclusion bodies in cells up to levels higher than half of the cell mass, which is a time-consuming process. For PHA production, a two-stage continuous cultivation system with two fermenters connected in series is a potentially useful system. It offers production of cells at a specific growth rate in a first compartment at conditions that lead cells to generate PHA at higher rates in a second compartment, with a relatively long residence time. In such a system, dilution rates of 0.21 h(-1) in the first fermentor (D-1) and 0.16 h(-1) in the second fermenter (D-2) were found to yield the highest volumetric PHA productivity. Transient-state experiments allowed investigation of D-1 and D-2 over a wide dilution rate range at high resolution in time-saving experiments. Furthermore, the influence of temperature, pH, nutrient limitation, and carbon source on PHA productivity was investigated and results similar to optimum conditions in single-stage chemostat cultivations of P. oleovorans we re found. With all culture parameters optimized, a volumetric PHA productivity of 1.06 g L-1 h(-1) was determined. Under these conditions, P. oleovorans cells contained 63% (dry weight) PHA in the effluent of the second fermenter. This is the highest PHA productivity and PHA content reported thus far for P. oleovorans cultures grown on alkanes. (C) 2001 John Wiley & Sons, Inc.