A 94,000-dalton protein that has been shown to modulate microtubule assembly in a Mg2+- or Ca2+-dependent manner (Nishida & Sakai (1980) J. Biochem. 88, 1577-1586) was here shown to inhibit actin polymerization. The protein factor inhibited the rate and the extent of actin polymerization under nearly physiological conditions (for example, in 3 mM MgCl2 plus 90 mM KCl at pH 6.8). The inhibitory effect was dependent on divalent cation concentration; the lower the Mg2+ concentration was, the weaker the inhibitory effect. The inhibition was stoichiometric; addition of the protein factor caused a linear decrease in the extent of actin polymerization as measured by the viscosity increase, and under optimal conditions for inhibition about an equimolar amount of the protein factor was sufficient to inhibit the actin polymerization completely. Furthermore, inclusion of the protein factor increased the critical concentration of actin required for polymerization by a concentration nearly equivalent to that of the added factor. These results suggest the formation of a 1 : 1 complex between actin and the protein factor, which does not polymerize at all. Another assay for actin polymerization, pelleting of actin filaments by ultracentrifugation, confirmed the inhibitory effect of the protein factor. In addition to the inhibitory effect on polymerization, the protein factor had the ability to depolymerize actin filaments. We have temporarily called this protein factor PI factor. It may play an important role in cell structure and function through its interactions with actin and microtubules.