Resistance to permethrin and the activity of metabolic enzymes were monitored in field-collected Culex tarsalis from northeastern Colorado during 2005 and 2006 and compared with a lab strain from Bakersfield, CA. Collections were made from 13 different sites within and outside of the mosquito abatement area. Resistance levels by year and early vs. late season were examined and median lethal time values and 95% confidence intervals were compared with the lab strain. Mosquitoes collected in 2005 were more resistant to permethrin than either the lab strain or mosquitoes collected in 2006. Glutathione S-transferase levels recorded in 2005 were 9–12× higher than either the lab strain or 2006 mosquitoes; activity of mixed-function oxidases was also greater. Both mechanisms may play a role in permethrin resistance or reflect nontarget exposure to other pesticides in Cx. tarsalis in northeastern Colorado.