Ovale malaria is caused by two closely related parasite species, Plasmodium ovale curtisi (Poc) and P. o. wallikeri (Pow). These have recently been shown to differ in the duration of the latent period prior to development of a symptomatic blood-stage infection, implying differences in liver stage biology. The literature contains a single study on liver-stage schizonts (1954) and none on hypnozoites of ovale malaria, yet recent data suggest that ovale parasites can evade chemotherapy in some individuals, presumably through the emergence of hypnozoites into the blood after drug levels have fallen. Thus P. ovale spp. liver-stage biology has important public health consequences for malaria control and elimination efforts in sub-Saharan Africa, where the parasite is widespread. We have established a transient 48 - 72 hour culture method with ex vivo ovale parasites from blood samples submitted to the UK Malaria Reference Laboratory that sustains schizont and gametocyte maturation, but not-erythrocyte invasion. This will permit studies of mRNA expression, sex ratio and ex-flagellation in ovale sexual stage parasites for the first time. We will attempt experimental mosquito infections and initiation of in vitro liver stage cultures by inoculation of primary human hepatocytes and cell lines with sporozoites from both Poc and Pow. The ex vivo blood stage material will also be used to for next-generation sequencing on the LSHTM in-house Illumina platform. Bioinformatic approaches will be used to identify genes shared with the relapsing parasite P. vivax, but not with P. knowlesi, as a means to generate a list of candidate genes important in the formation of hypnozoites. Patterns of ovale relapse in Malian children treated with ACT for falciparum malaria will be deduced from screening of follow-up blood samples in large clinical trials currently underway; this will add to our knowledge of the role of hypnozoites in maintaining ovale transmission in Africa.