The Water Framework Directive (WFD) establishes the need to define specific reference conditions for the assessment of river ecosystems. This paper presents a multimetric index for assessment of biological quality in a river ecosystem using benthic macroinvertebrates. The study area corresponds to the headwater of the Cabe River, Northwest Spain, upstream of the irrigation dam at Ribasaltas, a water catchment for irrigation in the Lemos Valley area. Because Lemos Valley is an important irrigation district located in an area with key environmental features, defining the quality objectives that must be used to assess the impact of agriculture becomes essential. In our research, reference stations were chosen on the basis of a number of strict conservation criteria, and the macroinvertebrate community present in those stations was analysed. Following the verification of the homogeneity of environmental features in the study area, we selected the biological indices and metrics that provided the strongest correlations between the condition of the macroinvertebrate community and the overall environmental quality. After the most suitable metrics were selected, the weight of each metric in the final multimetric index was determined on the basis of the greater or lesser ability of each metric to define biological quality. The correlation between biological metrics and quality, and the ability of the selected metrics to define quality were determined on the basis of their performance at a number of control stations with different levels of disturbance. Finally, the quality thresholds for the proposed multimetric index were established such that the quality thresholds of the index matched the quality levels defined in the WFD. Copyright © 2013 John Wiley & Sons, Ltd.