N2 fixation (C2H2 reduction) associated with the roots, rhizomes, and sediments (rhizosphere cores) of the seagrass Thalassia testudinum was measured at sites in South Florida (Soldier Key, Biscayne Bay) and the Bahamas (Bimini Harbor). Rates of C2H2 reduction were higher in anaerobic than in aerobic assays and were linear for several hours after an initial lag period of 1-2h. Nitrogenase activity was proportional to the weight of rhizomes plus roots but showed no correlation with the total weight of the rhizosphere cores. C2H2 reduction occurred to depths of at least 30 cm but the majority (<85%) of the activity was in the 0- to 20-cm fraction; also the ratio of activities for the 0- to 10- and 10- to 20-cm depths was about 2:1. Most investigations were carried out using anaerobic assays of the 0- to 10-cm fractions and rates calculated for the period of 3-6 h after adding C2H2. These rates were not stimulated by organic compounds (glucose, lactate, succinate) but were approximately halved by a decrease in temperature of 10 degrees C. In a seasonal study at Soldier Key the rates of N2 fixation varied about 20-fold with maximal rates in late summer and minimal rates in winter (January). On a diurnal basis, C2H2 reduction increased in the morning but was depressed in midafternoon, probably due to O2 buildup in the rhizosphere. Daily rates of N2 fixation, during the summer months of 1975-1978, varied between 5 and 24 mg N (-2)m and the estimated annual rates of N2 fixation were 10-50 kg N (-1)ha, taking into account seasonal variations and activities to a depth of 20 cm.