The small molecule inhibitor CHIR99021 (CHIR) is well known as a selective glycogen synthase kinase-3 inhibitor. The purpose of our study was to optimize the conditions of CHIR supplementation that will enhance the proliferation of human induced pluripotent stem cells (hiPSCs) while maintaining their undifferentiated state under feeder-free conditions in adherent cultures for 4 days. Our results revealed that both of the timing and concentration of CHIR affected cell behaviors of hiPSCs, such as colony formation, cell proliferation, and differentiation. The addition of 1-3 mu M CHIR to hiPSCs cultures in the late 2-day period of a 4-day cultivation was effective in enhancing cell proliferation. Treatment with 3 mu M CHIR significantly enhanced cell proliferation, but led to differentiation of hiPSCs when the treatment was carried out over 4 days. Treatment with higher concentration of CHIR was also conducive to deviating hiPSCs from their undifferentiated state. Treatment with 10 mu M CHIR led to decreased expression of pluripotency-associated genes and increased level of mesoderm marker genes, but failed to provided any growth-promoting effect. Interestingly, when treatment with 1 mu M CHIR was confined to the late 2-day period of a 4-day cultivation, cell proliferation was enhanced without detectable deviation from the undifferentiated state as evidenced by the expression levels of pluripotency-associated genes, e.g., OCT3/4, NANOG, SOX2, and REXI. Repeated use of 1 mu M CHIR in subcultures provided no adverse effect on the proliferation of hiPSCs. Our results indicated that carefully designed CHIR treatment allows for enhanced proliferation of hiPSCs. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.