BACKGROUND: Sulfadoxine-pyrimethamine (S/P) is widely used for treatment of failures following the first line treatment for malaria in Africa. In Guinea-Bissau, it has been recommended as second line therapy by the National Malaria Programme since 1996. In order to monitor any change of the in vivo sensitivity, the efficacy of S/P was studied immediately before the introduction of the drug and 6-9 years later. METHODS: Children participating in clinical in vivo studies were given S/P if having late clinical treatment failure following the treatment with quinine, chloroquine, or amodiaquine. Parasitological and clinical failures were evaluated during a 35-day follow-up. During the first study period whole blood sulfadoxine concentrations were measured on day 7. RESULTS: Altogether, 56 children failed the initial treatment and were included in 1995/1996 as well as 55 children in 2002/2004. The PCR-uncorrected adequate clinical and parasitological response rates on day 28 were 94% and 91%, and on day 35 they were 89% and 91%, respectively, in the two periods. No difference between median blood drug concentration in children with and without treatment failure was observed. INTERPRETATION: The efficacy of S/P as second line treatment for uncomplicated malaria has remained unchanged in spite of a relatively high level of genetic markers associated with Plasmodium falciparum resistance to S/P previously found in the area.