BACKGROUND: Poly-p-hydroxybutyrate (PHB), produced by several species of bacteria, has attracted great attention as a biodegradable and biocompatible compound with similar properties to polypropylene. Unfortunately, its use is currently limited due to high production costs. One of the most common methods for overcoming this constraint is the use of inexpensive substrates, like methanol, in high cell density cultivations (HCDC). RESULTS: Fermentation was carried out with optimized feed composition (639 g l(-1) methanol, 4 g l(-1) MgSO4.7H2O, 41 mL L-1 trace elements, 5.6 g L-1 NaH2PO4 center dot H2O and 24.3 g L-1 K2HPO4) and a feeding strategy based on the detection of substrate limitation by dissolved oxygen (DO). After 35 h, at which dry cell weight (DCW) reached a value of 70 g L-1, PHB production was stimulated, and biomass and PHB productivities of 2.8 and 0.98 g L-1 h(-1) were obtained, respectively. These results surpassed those reported in the literature for PHB production from methanol by Methylobacterium species. CONCLUSION: The proposed feed composition and feeding strategy for PHB production from methanol by Methylobacterium extorquens 1340 in fed-batch cultivation resulted in high biomass and PHB productivity. They can be implemented for recombinant bioproducts (proteins) produced by M. extorquens due to the lack of PHB accumulation in the growth phase. (C) 2009 Society of Chemical Industry