<Background> Sendai virus (SeV) is an enveloped virus with a negative-stranded RNA genome and is a member of the family Paramyxoviridae. SeV-based vectors replicate exclusively in the cytoplasm of transduced cells, and do not go through a DNA phase; therefore, there is no concern about the unwanted integration of foreign sequences into the host chromosomal DNA, which indeed caused a life-threatening disease in a few limited ceases.Human ES cells are expected to have clinical applications as well as to serve as a basic research tool. Foreign genes are often transferred into ES cells for genetic marking or regulating differentiation. We have previously shown that SeV vector can introduce a foreign gene efficiently and stably into cynomolgus monkey ES cells while preserving the pluripotency. The transgene expression, however, should be suppressed in cases of unexpected toxicity or when only a temporal transgene expression is necessary. Since SeV is an RNA virus, we have examined whether anti-RNA virus agents are able to remove the SeV vector from transduced ES cells.<Methods> The F-defective SeV vector carrying the GFP gene was constructed. Cynomolgus ES cells were transduced with the vector at 10 MOI per cell (transduction efficiency 60%). Fluorescent ES-cell colonies were plucked 1 month post-transduction and propagated in the regular FBS-supplemented medium (GFP-positive cells > 90%). The cells were treated with anti-viral agents which were reported to suppress the replication of SeV; ribavirin (12–20 μM), papaverine (1–100 μM), and/or prostaglandin A1 (PGA1; 0.3–30 μM). The regular culture medium was DMEM/F12 with 15% FBS. The serum-free culture medium was DMEM/F12 with 20% Knockout serum replacement (KSR, Invitrogen).<Results and Discussion> Although the GFP expression in ES cells was suppressed by treatment with either ribavirin or papaverine, the cells showed differentiated phenotypes. Treatment with PGA1 led to the apoptosis of ES cells at lower concentrations than those required to suppress the GFP expression. Treatment with a mixture of these three agents successfully suppressed the GFP expression in the SSEA-4-negative, differentiated population. On the other hand, cultivation of the transduced ES cells in the KSR-supplemented medium, a widely used serum-free recipe for ES cell cultures, resulted in the complete suppression of GFP expression in ES cells in the undifferentiated state. The SeV genome and GFP gene were undetectable even by RNA-PCR. KSR was originally developed to maintain ES cells in an undifferentiated state, and this is the first report about its unexpected antiviral activities. The anti-viral effect of KSR was cancelled by the addition of 15% FBS. KSR, however, had no effect on the SeV-mediated expression of GFP in LLC-MK2, a standard control cell line for SeV transduction. Unfortunately, information on the composition of KSR is not available, and we cannot explain the mechanism by which KSR eradicates the SeV genome.<Conclusions> Although none of the antiviral agents alone; ribavirin, papaverine, or PGA1, is effective at suppressing SeV vector-mediated transgene expression in ES cells, a mixture of the three agents may be useful for suppressing the transgene expression after the differentiation of the transduced ES cells. On the other hand, KSR should be useful in terms of eradicating the SeV genome from transduced ES cells as well as maintaining the pluripotency.