Oncolytic herpes simplex viruses (HSV) hold promise for therapy of glioblastoma multiforme (GBM) resistant to traditional therapies. We examined the ability of genetically engineered HSV to infect and kill cells that express CD133, a putative marker of glioma progenitor cells (GPC), to determine if GPC have an inherent therapeutic resistance to HSV. Expression of CD133 and CD111 (nectin-1), the major entry molecule for HSV, was variable in six human glioma xenografts, at initial disaggregation and after tissue culture. Importantly, both CD133+ and CD133- populations of glioma cells expressed CD111 in similar relative proportions in five xenografts, and CD133+ and CD133- glioma cell subpopulations were equally sensitive to killing in vitro by graded dilutions of wild-type HSV-1(F) or several different gamma(1)34.5-deleted viruses. GPC did not display an inherent resistance to HSV. While CD111 expression was an important factor for determining sensitivity of glioma cells to HSV oncolysis, it was not the only factor. Our findings support the notion that HSV will not be able to effectively enter, infect, and kill cells in tumors that have low CD111 expression (<20%). However, virotherapy with HSV may be very effective against CD111+ GPC resistant to traditional therapies.