The distribution of nitrogenase activity in the rice-soil system and the possible contribution of epiphytic cyanobacteria on rice plants and other macrophytes to this activity were studied in two locations in the rice fields of Valencia, Spain, in two consecutive crop seasons. The largest proportion of photodependent N 2 fixation was associated with the macrophyte Chara vulgaris in both years and at both locations. The nitrogen fixation rate associated with Chara always represented more than 45% of the global nitrogenase activity measured in the rice field. The estimated average N 2 fixation rate associated with Chara was 27.53 kg of N ha ؊1 crop ؊1 . The mean estimated N 2 fixation rates for the other parts of the system for all sampling periods were as follows: soil, 4.07 kg of N ha ؊1 crop ؊1 ; submerged parts of rice plants, 3.93 kg of N ha ؊1 crop ؊1 ; and roots, 0.28 kg of N ha ؊1 crop ؊1 . Micrographic studies revealed the presence of epiphytic cyanobacteria on the surface of Chara. Three-dimensional reconstructions by confocal scanning laser microscopy revealed no cyanobacterial cells inside the Chara structures. Quantification of epiphytic cyanobacteria by image analysis revealed that cyanobacteria were more abundant in nodes than in internodes (on average, cyanobacteria covered 8.4% ؎ 4.4% and 6.2% ؎ 5.0% of the surface area in the nodes and internodes, respectively). Epiphytic cyanobacteria were also quantified by using a fluorometer. This made it possible to discriminate which algal groups were the source of chlorophyll a. Chlorophyll a measurements confirmed that cyanobacteria were more abundant in nodes than in internodes (on average, the chlorophyll a concentrations were 17.2 ؎ 28.0 and 4.0 ؎ 3.8 g mg [dry weight] of Chara ؊1 in the nodes and internodes, respectively). These results indicate that this macrophyte, which is usually considered a weed in the context of rice cultivation, may help maintain soil N fertility in the rice field ecosystem.Rice fields are one of the most extensive freshwater ecosystems on Earth, covering about 150 ϫ 10 6 ha. In spite of the widespread dominance of rice plants, a conspicuous photosynthetic aquatic biomass composed of cyanobacteria, planktonic, filamentous, and macrophytic algae, and vascular macrophytes develops during different phases of rice growth and competes with rice for nutrients and light (23). Nitrogen is a major factor in rice production. However, the efficiency of N fertilizer is one of the lowest efficiencies of all plant nutrients due to large N losses from flooded soils (7). Native soil nitrogen is the main N source for rice, accounting for more than 50% of the N in the rice plant (9, 10, 17), and so long-term sustainability of rice cultivation depends on the use and effective management of internal resources that maintain soil N fertility. The soil N pool is believed to be maintained by biological nitrogen fixation (17,25). In fact, biological N 2 fixation has allowed a stable and moderate yield to be maintained in traditional low-input rice cul...