<ns4:p><ns4:bold>Background: </ns4:bold>Individuals living in malaria-endemic regions develop naturally acquired immunity against severe malarial disease, but it is unclear whether immunity that affects the establishment of infections develops following continuous natural exposure.</ns4:p><ns4:p> <ns4:bold>Methods: </ns4:bold>We cleared schoolchildren in Burkina Faso of possible sub-patent infections and examined them weekly for incident infections by PCR. Plasma samples collected at enrolment were used to quantify antibodies to the pre-eryhrocytic-stage antigens circumsporozoite protein (CSP) and liver stage antigen. Sporozoite gliding inhibition by naturally acquired antibodies was assessed using <ns4:italic>Plasmodium falciparum </ns4:italic>NF54 sporozoites; hepatocyte invasion was assessed using the human HC-04 hepatoma cell line and NF54 sporozoites. The associations between these functional pre-erythrocytic immunity phenotypes and time to PCR-detected infection were studied.</ns4:p><ns4:p> <ns4:bold>Results: </ns4:bold>A total of 51 children were monitored; the median time to first detection of infection by PCR or development of clinical symptoms was 28 days. Anti-CSP antibody titres showed a strong positive association with sporozoite gliding motility inhibition (P&lt;0.0001, Spearman’s ρ=0.76). <ns4:italic>In vitro </ns4:italic>hepatocyte invasion was inhibited by naturally acquired antibodies (median invasion inhibition, 19.4% [IQR 15.2-40.9%]), and there was a positive correlation between gliding and invasion inhibition (P=0.02, Spearman’s ρ=0.60). Survival analysis indicated longer time to infection in individuals displaying higher-than-median sporozoite gliding inhibition activity (P=0.01).</ns4:p><ns4:p> <ns4:bold>Conclusions: </ns4:bold>In summary, functional antibodies against the pre-erythrocytic stages of malaria infection are acquired in children who are repeatedly exposed to <ns4:italic>Plasmodium </ns4:italic>parasites. This immune response does not prevent them from becoming infected during a malaria transmission season, but might delay the appearance of blood stage parasitaemia and consequently needs to be considered in the evaluation of malaria vaccines.</ns4:p>