Azotobacter salinestris, a sodium-dependent. microaerophilic N-2-fixing soil bacterium, formed polyhydroxyalkanoate copolymers comprised of beta-hydroxybutyric acid and 9-12 mol% beta-hydroxyvaleric acid (HV) during growth on sugars. Increased HV content was achieved by feeding valeric acid to the culture growing on glucose, but propionic acid could be directed to HV formation only when it served as the sole C source. Polymer production in nitrogen-fixing cells was increased at higher aeration, provided that a complex organic nitrogen source was also present, but there was no HV in the polymer. HV production was increased to 28 mol% in nitrogen-fixing cells when aeration was lower and acetate was provided with glucose in the medium. Enzymes leading to the production of polyhydroxyalkanoate copolymers were found to be similar in A. salinestris and Azotobacter vinelandii, but A. vinelandii is unable to form HV from propionate or from sugars without valeric acid addition. A biochemical scheme is proposed for the production of HV in A. salinestris, whereby the glyoxylate bypass assimilates acetate to generate succinate, which may be converted into propionyl-CoA for HV synthesis. The results suggest that it may be possible to control the molar yield of HV formed from sugars by A. salinestris.