The characteristics of S-II, a factor stimulating partially purified RNA polymerase II, were studied further. It was found that S-II was inactivated by digestion with trypsin or heat-treatment at 60°C for lOmin, indicating that it consists at least partly of protein. No ribonuclease [EC 2.7.7.16] or deoxyribonuclease [EC 3.1.4.5] activity was detected in S-II, except ribonuclease H activity which was not separated from the stimulatory activity on a column of CM-cellulose. The ribonuclease H activity in the S-II preparation was repressed by MnCl2 without loss of stimulatory activity. Using Ehrlich ascites tumor DNA as template it was found that the molecular size of RNA was much larger when synthesized in the presence of S-II. However, using calf thymus DNA as template the molecular size of the RNA synthesized was not affected by S-II.