NEIL1, the mammalian homolog of Escherichia coli endonuclease VIII, is a DNA glycosylase that repairs ring-fragmented purines, saturated pyrimidines and several oxidative lesions like 5-hydroxyuracil, 5-hydroxycytosine, etc. Previous studies from our laboratory have shown that Werner Syndrome protein (WRN), one of the five human RecQ helicases, stimulates NEIL1 DNA glycosylase activity on oxidative DNA lesions. The goal of this study was to extend this observation and analyze the interaction between NEIL1 and all five human RecQ helicases. The DNA substrate specificity of the interaction between WRN and NEIL1 was also analyzed. The results indicate that WRN is the only human RecQ helicase that stimulates NEIL1 DNA glycosylase activity, and that this stimulation requires a double-stranded DNA substrate.