3-Mercaptolactate (3ML) is an interesting mercapto compound with special regard to the biosynthesis of new polythioesters (PTEs). Unfortunately, this thioester analog of lactic acid is currently not commercially available. For this reason, we developed an in vitro biosynthesis pathway to convert cysteine to 3-mercaptopyruvate (3MPy), which is then rapidly and efficiently converted to 3ML by suitable lactate dehydrogenases (LDHs). As liver LDH from Rattus norvegicus (LDHRn) was previously described to Exhibit 3MPy reduction activity, in silico studies based on homology to LDHRn were performed and led to the identification of four potentially suitable bacterial LDH candidates from Escherichia coli (LDHEc), Corynebacterium giutamicum (LDHcg), Bacillus cereus (LDHBc) and Gloeobacter violaceus (LDHGv). After heterologous expression in E. coil followed by purification, the enzymes were assessed for their potential to reduce 3MPy to 3ML in comparison to LDHRn. With 3MPy, LDHs from E. coli, C. glutamicum and B. cereus showed no or only very low specific activities of 0.23 +/- 0.1 U/mg (LDHcg) and 0.08 +/- 0.2 U/mg (LDHBc), respectively. In contrast, LDHG, exhibited a remarkable specific activity of 63.6 +/- 8.1 U/mg, being even twice as active as the R. norvegicus LDH. To verify LDH-catalyzed biosynthesis of 3ML we developed and optimized a detection method allowing qualitative analysis and quantification of 3MPy and 3ML by derivatization with Ellman's reagent and liquid chromatography-mass spectrometry. This study shows once more the impressive versatility of LDHs and presents a rapid and efficient biosynthesis process for 3ML, a biotechnologically interesting, yet hard-to-obtain, compound.