In this study, we have investigated the transcriptome of Ralstonia eutropha H16 during cultivation with gluconate in presence of 3,3'-thiodipropionic acid (TDP) or 3,3'-dithiodipropionic acid (DTDP) during biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptopropionate). Genome-wide transcriptome analyses revealed several genes which were upregulated during cultivation in presence of the above-mentioned compounds. Obtained data strongly suggest that two ABC-type transport system and three probable extracytoplasmic solute receptors mediate the uptake of TDP and DTDP, respectively. In addition, genes encoding the hydrolase S-adenosylhomocysteinase AhcY and the thiol-disulfide interchange proteins DsbA, DsbD, and FrnE were upregulated during cultivation on DTDP and, in case of AhcY and FrnE, on TDP as well. It is assumed that the corresponding enzymes are involved in the cleavage of TDP and DTDP. Several genes of the fatty acid metabolism exhibited increased expression levels: genes encoding two acetyltransferases, a predicted acyltransferase, the acetoacetyl-CoA reductase phaB3, an enoyl-CoA hydratase as well as an acyl dehydratase, an acetyl-CoA synthetase, two acyl-CoA dehydrogenases, the methylmalonyl-CoA mutase encoded by sbm1 and sbm2 and phaY1 were detected. Furthermore, ORF H16_A0217 encoding a hypothetical protein and exhibiting 54% amino acids identical to an acyl-CoA thioesterase from Saccharomonospora viridis was found to be highly upregulated. As the 2-methylcitrate synthase PrpC exhibited a three- to fourfold increased activity in cells grown in presence of TDP or DTDP as compared to gluconate, metabolization of the cleavage products 3MP and 3-hydroxypropionate to propionyl-CoA is proposed.