Existing data regarding alcohol ethoxylate (AE) surfactants indicate that structures with greater than 20 ethoxylate (EO) units per molecule or with multibranched alkyl chains may not pass a ready biodegradability test. This could have important consequences for complying with regional regulatory requirements and for the potential risks these chemicals could present to the environment. We investigated the influence of chemical structure on the biodegradability of AEs with different alkyl chain branching and EO content. The AEs investigated were a multibranched AE (average, 18 EO), an oxo-AE (monobranched; average, 23 EO), and a linear AE of oleochemical origin (average, 40 EO). The aims of the present study were to assess the ready biodegradability of AEs with high EO content and to establish the mechanism or pathway by which biodegradation occurs for the oxo-AE. Biodegradation studies were conducted using standard test conditions (International Standards Organization 14593). Solid-phase extraction and liquid chromatography with electrospray mass spectrometry were used to detect profiles in both derivatized and underivatized extracts of samples. Derivatization with phthalic anhydride was used to improve ionization of the lower-ethoxylated AEs and free alcohol that were key indicators in the present study. All AEs were rapidly biodegraded, achieving more than 60% mineralization of parent material. Central fission was the predominant mechanism for the oxo-AE, as confirmed by identification of the oligomeric distribution and quantification of polyethylene glycol released during biodegradation.