The aim of this retrospective study was to evaluate the Immunoquick+4 (BioSynex, Strasbourg, France), a three-band malaria rapid diagnostic test (MRDT) targeting histidine-rich protein-2 (HRP-2) and pan Plasmodium-specific parasite lactate dehydrogenase, in a non-endemic reference setting. Stored whole-blood samples (n = 613) from international travellers suspected of malaria were used, with microscopy corrected by polymerase chain reaction (PCR) as the reference method. Samples infected by P. falciparum (n = 323), P. vivax (n = 97), P. ovale (n = 73) and P. malariae (n = 25) were selected, as well as 95 malaria-negative samples. The overall sensitivities of the Immunoquick+4 for the diagnosis of P. falciparum, P. vivax, P. malariae and P. ovale were 88.9, 75.3, 56.0 and 19.2%, respectively. Sensitivity was significantly related to parasite density for P. falciparum (93.6% versus 71.4% at parasite densities >100/microl and <or=100/microl, respectively) and P. vivax (86.8% versus 48.3% at parasite densities >500/microl and <or=500/microl, respectively). The Immunoquick+4 showed good reproducibility and reliability for both test results and line intensities. The Immunoquick+4 performed well for the detection of P. falciparum and P. vivax.