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Mosquitoes of the Anopheles maculipennis Meigen ( Diptera: Culicidae) group are of public health concern: five of the 11 morphologically indistinct species have been historically considered as vectors of malaria in Europe. Three members of the An. maculipennis group have been reported in the U. K.: Anopheles atroparvus van Thiel; Anopheles messeae Falleroni, and Anopheles daciae Linton, Nicolescu & Harbach. To study the distribution of the three U.K. species, particularly that of An. daciae, we developed a polymerase chain reaction-Restriction fragment length polymorphism ( PCR-RFLP) assay using the nuclear ribosomal internal transcribed spacer 2 ( ITS-2) gene. Anopheles daciae was found to be widespread, occurring in four of the five counties surveyed in southern England and on the Welsh island of Anglesey, often in sympatry with the closely related species An. messeae. The host preferences of 237 blood-fed females were determined using either direct sequencing or PCR-based fragment analysis of the mitochondrial cytochrome oxidase b gene with DNA from females' abdomens. All three species were found to be opportunistic, having fed on at least three different hosts. Seventeen individuals contained multiple bloodmeals, including two An. daciae that had fed on humans and birds. Our results show that An. daciae is widespread in England and Wales, occurs in sympatry with other members of the An. maculipennis group, and feeds on humans, which suggests it is a potential vector of disease in the U.K. [ABSTRACT FROM AUTHOR]