The potential of the production of polyhydroxyalkanoates (PHA), consisting of medium-chain-length (MCL) hydroxyfatty acids (C5–C14), in recombinant Escherichia coli was investigated. E. coli mutants affected in fatty acid degradation and fatty acid de novo synthesis were employed. We established the functional expression of the Pseudomonas aeruginosa PHA synthase gene phaC1. The coding region of phaC1 was subcloned via PCR into vector pBluescript SK−. The resulting plasmid pBHR71 enabled functional expression of phaC1 under lac promoter control and conferred synthesis and accumulation of PHA to various strains of E. coli. PHA synthesis was analysed with respect to the carbon source in various E. coli fad and fab mutants. This study provided evidence that intermediates of the fatty acid β-oxidation can be directed to PHA synthesis and that 3-hydroxydecanoyl-CoA is the main substrate for PHA synthase PhaC1 from P. aeruginosa. The E. coli fadB mutant LS1298 containing plasmid pBHR71 and cultivated in LB medium containing 0.5% (w/v) decanoate revealed the strongest accumulation of PHA contributing to about 21% of the cellular dry weight, which was composed of 2.5 mol% 3-hydroxyhexanoate, 20 mol% 3-hydroxyoctanoate, 72.5 mol% 3-hydroxydecanoate and 5 mol% 3-hydroxydodecanoate.