β-Actinin was purified by Sephadex G-200 column chromatography, and this preparation showed the same activity in keeping the dispersed state of sonicated F-actin as the /J-actinin purified by repeated (NH4)2SO4 fractionation. However, the sedimentation coefficient was 4.5 S instead of 8S and the molecular weight was 62,000 instead of 140,000. The intrinsic viscosity was 0.05 dl/g. Helical content was 13–15%, estimated by the methods of optical rotary dispersion and circular dichroism. In the presence of β3-actinin, 70–100 S component of F-actin was changed into 40 S component as revealed in the sedimentation pattern. The dynamic rigidity of F-actin was greatly reduced by the addition of β-actinin, 5–10% by weight to F-actin, whereas viscosity was decreased down to 50% of the control value. The infinitely high value of viscosity at very low velocity gradients (−1) was remarkably dropped by )9-actinin. These facts clearly indicate that the inter-filamental interactions of F-actin in solution is inhibited by /9-actinin. Factors influencing the activity of /9-actinin in dispersing F-actin after sonic treatment were investigated. It was very active in the presence of 0.1 M KC1 between pH 5.5 and 9.5, and at pH 7.2 between 0.05 m and 0.2 m KC1. /3-Actinin was quickly inactivated at 60°C, and at 70–80°C it was completely denatured within 5 min. ATP favored the action of /3-actinin. £-Chloromercuribenzoate did not affect /3-actinin, although it alone shortened F-actin when sonicated. 8 M Urea inactivated /3-actinin. The effects of trypsin [EC 3. 4. 4. 4], chymotrypsin [EX?- 3. 4. 4. 5], papain [EC 3.4.4.10] and Nagarse [EC 3.4.4.16] on polymerizability of G-actin and on particle length of F-actin were examined to see if partially digested actin be active as /3-actinin. F-actin partially digested by Nagarse showed a similar behavior after sonic treatment in the flow birefringence properties to the case with /3-actinin. But it turned out that the Nagarse-treated F-actin particles were randomly aggregated under the electron microscope. With the information of the localization of /3-actinin in the I band of myofibrils, the physiological role of /3-actinin is discussed.