Abstract The Ca 2+- and Mg 2+-ATP-phosphohydrolase (ATPase) activities measurable in suspensions of and extracts from bull epididymal spermatozoa flagella, sonicated for 2 min at 8 W, were compared with those of control flagella after 0, 3, and 18 h dialysis against an alkaline 0.5 M KCl extracting solution. Activity was measured in the absence of and in the presence of oligomycin. The effects of sonication and extraction on structural components within the flagella were visualized electron microscopically. Sonication caused fragmentation of flagella, extensive disruption of mitochondria and an immediate (0 h) increase in both Ca 2+- and Mg 2+-ATP-ase activity. Prolonged dialysis resulted in solubilization of specific flagellar structures, partial disruption of mitochondrial integrity, and increases in ATPase activity. Mg 2+-ATPases of flagellar suspensions and extracts were greater than Ca 2+-ATPases, and a part of this Mg 2+-ATPase activity was inhibited by oligomycin. Therefore, Mg 2+-ATPases from disrupted mitochondria contribute to the Mg 2+-ATPase activities measurable in suspensions of and extracts from bull sperm flagella. This study emphasizes the necessity of evaluating the effects of the mechanical and chemical treatments used in fractionating cells before interpreting the biochemical information derived from their isolated components.