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To better understand the mechanism of actin filament (F-actin) bundling by polyamines, we have measured the onset of bundling as a function of polyamine concentration. Samples were centrifuged at low speeds to separate bundles from unbundled actin, and the relative amounts of actin in the pellet and supernatant were determined via gel electrophoresis, yielding a description of the bundling transition as a function of actin and polyamine concentrations. These experiments were carried out for two different polyamines, spermine (tetravalent) and spermidine (trivalent). We found that the threshold concentration of polyamine needed to bundle actin is independent of both actin concentration and Mg2+ concentration over a wide range in Mg2+ concentration. We also find that spermine in F-actin bundles is essentially invisible in solution-phase proton NMR, suggesting that it is bound so tightly to F-actin that it is immobilized.