Halomonas marina HMA 103 (MTCC 8968), the moderately halophilic bacterium isolated and characterized from the solar saltern of Orissa, India, grows optimally at 10% (w/v) NaCl in culture medium and is able to synthesize poly(3-hydroxybutyrate) [P(3HB)] during growth. This study is an attempt to optimize the cultural conditions for efficient production of P(3HB) by H. marina in batch cultivation. Growth of the organism under shake-flask culture using 2% (w/v) glucose resulted in P(3HB) accumulation accounting for more than 59% of cell dry weight after 50 h of incubation. The optimum P(3HB) production was attained with a combined supply of NH <sub>4</sub> Cl and yeast extract as N-source, 0.01% (w/v) phosphate, 1.5% (w/v) sulphate and 10% (w/v) NaCl. Qualitative and quantitative <sup>1</sup> HNMR and FT-IR analysis of cells grown in alkanoic acids (C 3 -C 6 ) as sole source of carbon and co-substrates revealed synthesis of PHA co-polymers composed of 3-hydroxybutyric acid and 3-hydroxyvaleric acid [P(3HB-co-3HV)]. In two-step cultivation, accumulation of the co-polymer was significantly improved (80% CDW) in glucose medium supplemented with valerate (0.1%, w/v) as co-substrate and the polymer contained 88.1 and 12.8 mol% 3HB and 3HV monomers, respectively. © 2009 Akadémiai Kiadó, Budapest.