A simple device for marking phlebotomine sand flies with fluorescent powders is described and tested; the design of the new device is characterized by separate compartments for sand flies and powder. The effect of fluorescent powder on survival and mobility of the sand fly Lutzomyia longipalpis was compared using our device (method A) and a single-container method (method B). Mortality within 1 h of powder application was negligible for method A (0.9%), but was 19.8% for method B; in addition, method B also reduced sand fly mobility. Adherence of excess powder to the sand flies observed with method B was responsible for the negative effects observed during the marking process. In field releases, however, recapture rates were the same for each method. Neither sand fly mobility or survival were adversely affected if appropriate quantities (method A) of fluorescent powder were applied to the exoskeleton of these insects.