Alcohol ethoxylates (AEs) are surfactants widely used for domestic and industrial uses. Very little is known about the degradation pathway of BAEs, and structures of their related intermediates have not yet been fully characterized. We have used liquid chromatography-mass spectrometry(LC-MS) with an electrospray (ES) interface to fill in this gap. An example of BAEs, i.e., a 2-butyl branched A(12)E mixture with an average of five ethoxy units, was submitted to degradation, according to an OECD bioassay. After solid-phase extraction from samples of the bioassay solution taken at intervals, analytes were analyzed by LC-ES-MS. Elucidation of the metabolite structures were obtained by in-source collision-induced decomposition spectra. Initially, bacterial attack on the ethoxy chain (E-chain) produced species with the E-chain either shortened or, to a lesser extent, oxidized. Next, the major route for degradation produced abundant amounts of two new classes of rather stable intermediates via the omega, beta-oxidation mechanism of the main alkyl chain. At the same time, a minor biodegradation route was that of the simultaneous attack of both alkyl chains by the omega, beta-oxidation process. This route produced species which were rapidly converted by E-chain oxidation and beta-oxidation mechanisms to very polar intermediates. After a rather long bacteria acclimation time, the major route was reactivated with shortening of the alkyl side chain via the omega,beta-oxidation mechanism and the same intermediates as those formed through the minor route were produced.