The distribution and ecology of mosquitoes of the Anopheles maculipennis complex were studied in the delta of the rivers Rhine and Meuse in the southwest of The Netherlands. The study area was previously malarious, with A. atroparvus being the only vector. 125 potential aquatic habitats of A. maculipennis were sampled, of which 47 (37.6%) contained larvae of this species complex. Larval densities varied from 7.4–325.93 larvae m−2. There was no correlation between chlorinity (‰) of the water and presence and/or density of larvae. The presence of A. maculipennis was not associated with one particular aquatic floristic habitat, although larvae were often found together with floating algae (Enteromorpha spp.). Larvae were not found in areas experiencing tidal flooding. A newly developed PCR method was used for identification of the mosquito sibling species. Of 150 larvae examined, only 4 were identified as A. atroparvus. All other larvae examined were A. messeae. Adult mosquitoes were identified as A. messeae and 30 wild-caught mosquitoes had fed on domestic animals. Because most anophelines found in 1999 were A. messeae, it is concluded that the study area has undergone a dramatic ecological change since the previous anopheline investigations in 1935, causing the near extinction of A. atroparvus. This species was the only malaria vector in The Netherlands and therefore it is not expected that malaria can return to its former endemic status in the coastal areas of The Netherlands.