With the increasing concern of potential loss of transgenic mosquitoes which are candidates as new tools for mosquito-borne disease control, methods for cryopreservation are actively under investigation. Methods to cryopreserve Anopheles gambiae sperm have recently been developed, but there are no artificial insemination or in vitro fertilization tools available. As a step to achieve this, we sought to identify a suitable medium for in vitro incubation of An. gambiae sperm and to tease out critical components that are involved in the sperm motility activation process. Using two cell viability assays, we identified the Biggers-Whitten-Whittingham (BWW) medium as suitable for in vitro incubation of An. gambiae sperm isolated from testes. We then modified the medium for motility assays by testing different HCO