The recent results of the Isotope Separation System (ISS) operations at the Tritium Systems Test Assembly (TSTA) with 100 g of tritium indicate that the system generally satisfies design goals, while system stability problems remain to be solved. We configured the ISS system for the three column mode, which is one of the promising cascade configurations in a fusion fuel cycle, to eliminate such instability and operated it for six days. Fluctuations in flows and liquid levels were improved. Column separation characteristics obtained were satisfactory and agreed with the numerical analysis. The amount of discharged tritium was an acceptable effluent level. This means that the existing ISS system can be used as a three column system and possibly be applied to numerous fuel concepts. Presently, a new laser Raman spectroscopic gas analyzer has been installed at the ISS. This on-line system enables studies of the ISS dynamic behavior for further stability and performance data.