The self-aggregation behavior of casein kinase II from Drosophila melanogaster has been analyzed by velocity sedimentation and electron microscopy. The results indicate that self-aggregation involves the formation of linear polymers or filaments approximately 10 nm in diameter. In the presence of 1 mM EDTA filament length was inversely proportional to total ionic strength over a range from 0.05 to 0.28, and filaments as long as 0.5 micron were observed at the lower ionic strengths. Similar results were obtained in the presence of 10 mM MgCl2, but two additional ionic strength-dependent phenomena were superimposed. First, at subphysiological ionic strength side-to-side aggregation of filaments occurred which resulted in enzyme precipitation. Second, at physiological ionic strength a time- and temperature-dependent increase in filament length occurred which generated polymers up to 5 micron long. No side-to-side aggregation occurred under the latter conditions. Filamentous forms of the kinase could be readily reconverted to the standard alpha 2 beta 2 tetramer by the addition of high salt. Filamentous casein kinase II was observed over a pH range from 6.8 to 8.0, at enzyme concentrations ranging from 6 to 150 micrograms/ml, in the presence of ATP, and at MgCl2 concentrations from 1 to 10 mM. However, time-dependent growth of long filaments was not observed at Mg2+ concentrations below 10 mM. The conditions under which filaments are observed in vitro suggest that they may also exist in vivo. The possibility that filament formation plays a role in the regulation of casein kinase II activity is discussed.