The present study was undertaken to analyze the resistance status of Culex pipiens pipiens (C. p. pipiens) to the chlorpyrifos insecticide in Tunisia. Three mosquito populations of C. p. pipiens were collected from three districts of Tunisia between 2003 and 2005. Results obtained here showed that all populations were resistant to used insecticide and the level of resistance varied widely between the samples. The resistance ratios at LC₅₀ (RR₅₀) to chlorpyrifos ranged from 8.75 to 455. A synergism study showed that resistance to chlorpyrifos was affected (SR₅₀ > 1, p < 0.05) by piperonyl butoxide (Pb), indicating the involvement of P450 oxidases detoxication in the recorded resistance of sample #1. However, esterases and/or glutathione S-transferase (GST)-mediated detoxication were not involved. Bioassays showed that resistance to chlorpyrifos and propoxur was correlated, suggesting an insensitive acetylcholinesterase (AChE 1). The highest frequencies of the resistant phenotypes ([RS] and [RR]) (0.74) were detected in the most resistant sample (#1). Five esterases were detected in the studied field samples. The esterase C1 encoded by the Est–1 locus and four esterases encoded by the Ester super locus: A1, A2–B2, A4–B4 (or A5–B5, which has the same electrophoretic mobility) and B12.