By means of affinity chromatography RNA-agarose a partial purification of a soluble TMV replicase is obtained in addition to a poly(U) polymerase when extracts of fresh, systemically TMV-infected tobacco leaves are used. An active replicase fraction always shows a certain amount of poly(U) polymerase. The replicase is inactivated by electrophoresis and the enzyme shows a harmful adsorption to certain supporting materials. When a purified replicase fraction, or an extract from frozen infected leaves, was gel filtrated on Sephadex G-75 in 50 mM Tris-HC1 pH 7.2, three peaks of replicase activity could be detected. Peak I was found in the void volume, peak II was elute with about 1.8 times the void volume and peak III with about 2.6 times the void volume. The activities of peaks II and III had very short half-times. Peak II was not obtained with extracts of frozen healthy leaves.