At temperate latitudes, Culex (Diptera: Culicidae) mosquitoes typically overwinter as adult females in reproductive arrest and also may serve as reservoir hosts for arboviruses when cold temperatures arrest viral replication. To evaluate their role in the persistence of West Nile virus (WNV) in the Sacramento Valley of California, the induction and termination of diapause were investigated for members of the Culex pipiens (L.) complex, Culex tarsalis Coquillett, and Culex stigmatosoma Dyar under field, seminatural, and experimental conditions. All Culex spp. remained vagile throughout winter, enabling the collection of 3,174 females and 1,706 males from diverse habitats during the winters of 2010–2012. Overwintering strategies included both quiescence and diapause. In addition, Cx. pipiens form molestus Forskäl females remained reproductively active in both underground and aboveground habitats. Some blood-fed, gravid, and parous Cx. tarsalis and Cx. pipiens complex females were collected throughout the winter period. Under both field and experimental conditions, Cx. tarsalis and Cx. stigmatosoma females exposed to autumnal conditions arrested primary follicular maturation at previtellogenic stage I, with primary to secondary follicular ratios <1.5 (indicative of a hormonally induced diapause). In contrast, most Cx. pipiens complex females did not enter reproductive diapause and ovarian follicles matured to ≥stage I–II (host-seeking arrest) or were found in various stages of degeneration. Diapause was initiated in the majority of Cx. tarsalis and Cx. stigmatosoma females by mid-late October and was terminated after the winter solstice, but hostseeking seemed limited by temperature. An accrual of 97.52 ± 30.7 and 162.85 ± 79.3 degree-days after the winter solstice was estimated to be necessary for diapause termination in Cx. tarsalis under field and seminatural conditions, respectively. An increase in the proportion of blood-fed Culex females in resting collections occurred concurrently with diapause termination in field populations based on ovarian morphometrics. WNV RNA was detected in one pool of 18 males and in a single blood-fed female Cx. tarsalis collected during winter. Therefore, both vertically and horizontally infected Culex females may persist through winter and possibly transmit WNV after diapause termination in late winter or early spring in the Sacramento Valley of California.