BACKGROUND: Polyhydroxyalkanoates (PHA) containing 3-hydroxy-4-methylvalerate (3H4MV) have better processing properties than other PHAtypes andwere not prone to ageing. Themain challenge in the production of these polymers is to simultaneously achieve high cell biomass, high polymer content, andhigh molar fraction of3H4MV. The aim of this studywas toinvestigate the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxy-4-methylvalerate) [P(3HB-co-3 HV-co-3H4MV)] by Cupriavidus eutrophus B-10646, synthesize polymers with different contents of 3H4MV and investigate their properties. RESULTS: It was shown that 4-methylvalerate (4MV, the precursor substrate) concentration in the culture medium must not be higher than 1 g L−1, and that4MVmust be carefully dosed, with at least 10–12 h intervals between supplementations.Aseries of polymers with molar fractions of 3H4MVbetween 2.7 and 11.3mol%was synthesized. Degrees of crystallinity, molecularweight characteristics, and thermal properties of the polymers have been investigated as dependent on proportions ofmonomers. CONCLUSION: The content of 3H4MV depends on the concentration of 4MV and conditions of carbon nutrition. The molar fraction of 3H4MV had a strong effect on the crystalline to amorphous region ratio. PHA containing 3H4MV specimens synthesized in this study had a degree of crystallinity of below 50%, which remained unchanged for 2.5 years.