Mediterranean streams are characterized by water flow changes caused by floods and droughts. When intermittency occurs in river ecosystems, hydrologic connectivity is interrupted and this affects benthic, hyporheic and flowing water compartments. Organic matter use and transport can be particularly affected during the transition from wet to dry and dry to wet conditions. In order to characterize the changes in benthic organic matter quantity and quality throughout a drying and rewetting process, organic matter, and enzyme activities were analyzed in the benthic accumulated material (biofilms growing on rocks and cobbles, leaves, and sand) and in flowing water (dissolved and particulate fractions). The total polysaccharide, amino acid, and lipid content in the benthic organic matter were on average higher in the drying period than in the rewetting period. However, during the drying period, peptide availability decreased, as indicated by decreases in leucine aminopeptidase activity, as well as amino acid content in the water and benthic material, except leaves; while polysaccharides were actively used, as indicated by an increase in b-glucosidase activity in the benthic substrata and an increase in polysaccharide content of the particulate water fraction and in leaf material. During this process, microbial heterotrophs were constrained to use the organic matter source of the lowest quality (polysaccharides, providing only C), since peptides (providing N and C) were no longer available. During the flow recovery phase, the microbial community rapidly recovered, suggesting the use of refuges and/or adaptation to desiccation during the previous drought period. The scouring during rewetting was responsible for the mobilization of the streambed and loss of benthic material, and the increase in high quality organic matter in transport (at that moment, polysaccharides and amino acids accounted for 30% of the total DOC). The dynamics of progressive and gradual drought effects, as well as the fast recovery after rewetting, might be affected by the interaction of the individual dynamics of each benthic substratum: sand sediments and leaves providing refuge for microorganisms and organic matter storage, while on cobbles, an active bacterial community is developed in the rewetting. Since global climate change may favor a higher intensity and frequency of droughts in