Summary objective To measure pyrethroid susceptibility in populations of malaria vectors and nuisance-biting mosquitoes in Tanzania and to test the biological efficacy of current insecticide formulations used for net treatment. methods Anopheles gambiae Giles s.l., An. funestus Giles s.l. and Culex quinquefasciatus Say were collected during three national surveys and two insecticide-treated net (ITN) studies in Tanzania. Knockdown effect and mortality were measured in standard WHO susceptibility tests and ball-frame bio-efficacy tests. Test results from 1999 to 2004 were compared to determine trends in resistance development. results Anopheles gambiae s.l. and An. funestus s.l. were highly susceptible to permethrin (range 87–100%) and deltamethrin (consistently 100%) in WHO tests in 1999 and 2004, while Culex quinquefasciatus susceptibility to these pyrethroids was much lower (range 7–100% and 0–84% respectively). Efficacy of pyrethroid-treated nets was similarly high against An. gambiae s.l. and An. Funestus s.l. (range 82–100%) while efficacy against Cx. quinquefasciatus was considerably lower (range 2–100%). There was no indication of development of resistance in populations of An. gambiae s.l. or An. funestus s.l. where ITNs have been extensively used; however, susceptibility of nuisance-biting Cx. quinquefasciatus mosquitoes declined in some areas between 1999 and 2004. conclusion The sustained pyrethroid susceptibility of malaria vectors in Tanzania is encouraging for successful malaria control with ITNs. Continued monitoring is essential to ensure early resistance detection, particularly in areas with heavy agricultural or public health use of insecticides where resistance is likely to develop. Widespread low susceptibility of nuisance-biting Culex mosquitoes to ITNs raises concern for user acceptance of nets.