Anion-exchange chromatography of crude extracts from the green alga Monoraphidium braunii yielded two glutamine synthetase (GS) activities. The ratio of activities was markedly different when crude extracts were subjected to various processing conditions but was not influenced by environmental factors of cell cultures. However, high performance liquid chromatography anion-exchange chromatograms showed only one GS if the crude extracts were processed immediately after cell disruption. Moreover, standard chromatography of crude extracts obtained in the absence of dithioerythritol, a reductant generally used in disruption buffers, yielded a single activity peak. Enzyme samples from the two activities obtained in the presence of dithioerythritol were purified for physicochemical characterization and antibody production. Both enzyme samples exhibited similar reactions to different inactivating agents and were undistinguishable by size-exclusion chromatography and native polyacrylamide gel electrophoresis. Additionally, the two GS preparations showed absolute antigenic identity as demonstrated by immunodiffusion and immunoblotting experiments. Immunocytochemistry of M. braunii cryosections evidenced a chloroplast-specific distribution of the enzyme, which rules out the existence of a cytoplasmic counterpart. All these results support the proposal that M. braunii possesses only one form of GS.