Publisher Summary This chapter describes the assay and purification of cytosolic factor (CF) required for cholera toxin activity. A cytosolic protein widespread in vertebrates is required in addition to a guanyl nucleotide triphosphate for the formation of a membrane complex that raises the ADP-ribosyl transferase activity of cholera toxin. Cool the testes of freshly slaughtered bulls on ice and conduct all subsequent steps of the purification at 0-4°. The purification can be improved by analyzing fractions by sodium dodecyl sulfate (SDS)-polyacrylamide minigels and selecting and pooling those fractions that minimize protein complexity. Pigeon erythrocyte membranes are particularly dependent on CF. Membranes can be prepared in bulk and stored until needed. Because crude CF often demonstrates inhibition as well as activation, it is important to use several concentrations of the unknown and to compare rising phases of the response curves. The recoveries from step to step are difficult to quantitate because CF of different purities gives response curves of different shapes and different plateaus.