Abstract Synthesis of polyhydroxyalkanoates (PHAs) by a new strain of Cupriavidus – Cupriavidus eutrophus B-10646 – was investigated under autotrophic growth conditions. Under chemostat, at the specific flow rate D=0.1h−1, on sole carbon substrate (CO2), with nitrogen, sulfur, phosphorus, potassium, and manganese used as growth limiting elements, the highest poly(3-hydroxybutyrate) [P(3HB)] yields were obtained under nitrogen deficiency. In batch autotrophic culture, in the fermenter with oxygen mass transfer coefficient 0.460h−1, P(3HB) yields reached 85% of dry cell weight (DCW) and DCW reached 50g/l. Concentrations of supplementary PHA precursor substrates (valerate, hexanoate, γ-butyrolactone) and culture conditions were varied to produce, for the first time under autotrophic growth conditions, PHA ter- and tetra-polymers with widely varying major fractions of 3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxyvalerate, and 3-hydroxyhexanoate monomer units. Investigation of the high-purity PHA specimens showed significant differences in their physicochemical and physicomechanical properties.