Gas-vacuolate filaments of Oscillatoria agardhii form a metalimnetic layer in Deming Lake, Minnesota. The environmental factors which affect buoyancy and the physiological processes which mediate changes in buoyancy were determined. Buoyant filaments lost their buoyancy in a few hours when incubated at light intensities above those found in situ (approximately 15 mumol photons m-2 s-1, or 1% of the surface value). The rate of buoyancy loss was accelerated by the addition of 10 muM phosphate at irradiances >200 mumol photons m-2 s-1. The effect of nutrient additions on buoyancy was also investigated over a longer time period by incubating metalimnetic samples in situ. The samples were deployed for 6 days at a depth where the irradiance was 8% of the surface value. As found in short-term experiments, the addition of phosphate resulted in the largest decrease in buoyancy. However, the addition of ammonia in addition to phosphate attenuated the buoyancy loss on day 2, and on day 6 the filaments in these treatments were almost completely buoyant. The physiological status of the filaments in these treatments was assayed by analysis of elemental ratios of C, N and P, and by measurement of cellular chlorophyll, polysaccharide and protein. In addition, the cellular content of gas vesicles was determined. The construction of ballast balance sheets from these data indicated that changes in buoyancy were primarily due to differences in the amount of polysaccharide ballast in the cells. However, in another set of in situ experiments, the increase in measured ballast molecules did not explain the observed loss of buoyancy. We hypothesized that another, undetected ballast-providing molecule had accumulated in the cells.