The objective of this work focuses on determining whether there are tumor cells with the Side Population phenotype in cell lines derived from astrocytomas, and if they are sensitive to hypoxia conditions and to the combination of temozolomide with inhibitors of sonic hedgehog pathway (cyclopamine) and of MGMT (O6 -benzylguanine). Cytometry studies were performed to separate the SP cell fraction in all cell lines. The proportion of SP cells was directly proportional to the degree of malignancy of the astrocytoma. The cells showed characteristics of tumor stem cells, such as a greater capacity for self-renewal, and constituted an independent or partially overlapping population with the population of CD133+ cancer stem cells. SP cells were highly resistant to temozolomide, while tumor cells that did not display tumor stem cell properties appeared to be more sensitive. The sonic hedgehog pathway caused resistance to temozolomide, but its inhibition with cyclopamine increased the cytotoxic effects of temozolomide, preferentially in populations not enriched for tumor stem cells of the Side Population. Chemoresistance was often independent of MGMT expression. O6 -benzylguanine was not always capable of increasing the sensitivity to temozolomide in tumor stem cells of the Side Population and in hypoxia.