The composition of microbial communities degrading trimethylolpropaneoleate, a synthetic fatty acid ester, was studied under conditions similar to a frequently used degradation test. A combination of conventional phenotypic tests and molecular biological methods, internal rRNA gene spacer amplification and randomly amplified DNA assay, was used for strain monitoring. Few bacterial species were selected from the large diversity of the inocula during degradation in the batch cultures. Growth depended on activity of extracellular lipases in the bacterial community. However, the enzyme producers made up only a minority. The dominating bacteria were not able to hydrolyze the synthetic ester in pure culture.