Cervical cancer is the third most frequent cancer in women worldwide. Adenine nucleotide signaling is modulated by the ectonucleotidases that act in sequence, forming an enzymatic cascade. Considering the relationship between the purinergic signaling and cancer, we studied the E-NTPDases, ecto-5'-nucleotidase, and E-NPPs in human cervical cancer cell lines and keratinocytes. We evaluated the expression profiles of these enzymes using RT-PCR and quantitative real-time PCR analysis. The activities of these enzymes were examined using ATP, ADP, AMP, and p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as substrate, in a colorimetric assay. The extracellular adenine nucleotide hydrolysis was estimated by HPLC analysis. The hydrolysis of all substrates exhibited a linear pattern and these activities were cation-dependent. An interesting difference in the degradation rate was observed between cervical cancer cell lines SiHa, HeLa, and C33A and normal imortalized keratinocytes, HaCaT cells. The mRNA of ecto-5'-nucleotidase, E-NTPDases 5 and 6 were detectable in all cell lines, and the dominant gene expressed was the Entpd 5 enzyme, in SiHa cell line (HPV16 positive). In accordance with this result, a higher hydrolysis activity for UDP and GDP nucleotides was observed in the supernatant of the SiHa cells. Both normal and cancer cells presented activity and mRNAs of members of the NPP family. Considering that these enzymes exert an important catalytic activity, controlling purinergic nucleotide concentrations in tumors, the presence of ectonucleotidases in cervical cancer cells can be important to regulate the levels of extracellular adenine nucleotides, limiting their effects.