BACKGROUND: The main aims of this study were to quantify the annual risk of falciparum malaria among nonimmune, UK-based airline crew and to undertake a risk assessment of short layovers in sub-Saharan African cities. METHODS: The number of nights exposed in malaria-endemic regions and reported cases of falciparum malaria were used to estimate annual disease incidence. Transmission risk estimates were calculated for each layover, that is, where crew were accommodated overnight, for one or more nights, in designated city hotels. Air-conditioning of ground transport, hotels, and airports was provided at all crew layovers. Details of activities associated with a risk for malaria and of adherence to antimosquito measures among crew were collected through a self-administered postal questionnaire. RESULTS: The annual risk of falciparum malaria was calculated to be 1.6 cases per 100,000 nights of exposure (95% CI 0.5-3.7). Crew reported widespread use of personal protection measures during the evenings when at risk. CONCLUSIONS: Attack rates of falciparum malaria were considerably lower than those reported in tourists during visits to sub-Saharan Africa. Factors contributing to this low attack rate included risk awareness, a protected sleeping environment, an urban setting, vector environmental controls, brief exposure, and good compliance with personal protection measures. Previously reported chemoprophylaxis compliance of < 10% in the same population was unlikely to have contributed to the low rate of disease. The longer layovers, of 3 or 4 nights, in some East African rosters provided greater opportunity for discretionary leisure activity away from protected hotel environments, and this needs to be considered in a risk assessment.