A study has been made of the presence and reactivity of the most commonly used surfactants, both anionic (linear alkylbenzene sulfonates, LAS, and alkyl ethoxysulfates, AES) and non-ionic (alcohol polyethoxylates, AEOs, and nonylphenol polyethoxylates, NPEOs), in water and surface sediments from the middle stretch of the Guadalete River in SW Spain (12 stations). Average values were between 0.1 and 3.7 mg kg⁻¹ in sediment, and between 0.2 and 37 μg L⁻¹ in water. The sorption of surfactants was dominated by hydrophobic mechanisms, so those homologues having longer alkyl chains (e.g. C₁₈AEO) showed higher relative percentages and concentrations in sediments compared with water. Local and sharply higher concentrations of these compounds were observed at three sampling stations (7, 9 and 12), indicating the occurrence of wastewater discharges into the river. By analysing the distributions of different surfactant homologues and their metabolites we were able to distinguish between sewage contamination from sources discharging treated and untreated wastewaters. Upstream (stations 1–2), LAS concentrations were below 30 μg L⁻¹ and the composition of their degradation intermediates (sulfophenyl carboxylic acids, SPCs) (160 μg L⁻¹) was dominated by short-chain homologues (C₆–C₉SPCs), indicating that the degradation of this surfactant is at an advanced stage. The highest concentration (487 μg L⁻¹) of SPCs was detected near the effluent outlet of a sewage treatment plant (STP) (station 12). Sampling stations (7 and 9) affected by untreated wastewater discharges were the only ones showing the presence of the most reactive and biodegradable SPC isomers and homologues (e.g. C₁₁SPC). Here, LAS reached the highest concentration values measured (>2 mg L⁻¹), and showed a homologue distribution closer to that of commercial mixtures than LAS found at the other stations.