Two peaks of glutamine synthetase (GS) activity were resolved by anion-exchange chromatography from the marine diatom Skeletonema costatum Grev. The second peak of activity accounted for greater than 93% of total enzyme activity, and this isoform was purified over 200-fold. Results from denaturing gel electrophoresis and gel-filtration chromatography suggest that six 70-kD subunits constitute the 400-kD native enzyme. The structure of the diatom GS, therefore, appears more similar to that of a type found in bacteria than to the type common among other eukaryotes. Apparent Michaelis constant values were 0.7 mM for NH4(+), 5.7 mM for glutamic acid, and 0.5 mM for ATP. Enzyme activity was inhibited by serine, alanine, glycine, phosphinothricin, and methionine sulfoximine. Polyclonal antiserum raised against the purified enzyme localized a single polypeptide on western blots of S. costatum cell lysates and recognized the denatured, native enzyme. Western analysis of the two peak fractions derived from anion-exchange chromatography demonstrated that the 70-kD protein was present only in the later eluting peak of enzyme activity. This form of GS does not appear to be unique to S. costatum, since the antiserum recognized a similar-sized protein in cell lysates of other chromophytic algae.