BACKGROUND: Controlled human malaria infection (CHMI) by mosquito bite is a powerful tool for evaluation of vaccines and drugs against Plasmodium falciparum malaria. However, only a small number of research centres have the facilities required to perform such studies. CHMI by needle and syringe could help to accelerate the development of anti-malaria interventions by enabling centres worldwide to employ CHMI. METHODS: An open-label CHMI study was performed with aseptic, purified, cryopreserved P. falciparum sporozoites (PfSPZ Challenge) in 36 malaria naive volunteers. In part A, the effect of the inoculation volume was assessed: 18 participants were injected intramuscularly (IM) with a dose of 2,500 PfSPZ divided into two injections of 10 microL (n = 6), 50 microL (n = 6) or 250 microL (n = 6), respectively. In part B, the injection volume that resulted in highest infectivity rates in part A (10 microL) was used to formulate IM doses of 25,000 PfSPZ (n = 6) and 75,000 PfSPZ (n = 6) divided into two 10-microL injections. Results from a parallel trial led to the decision to add a positive control group (n = 6), each volunteer receiving 3,200 PfSPZ in a single 500-microL injection by direct venous inoculation (DVI). RESULTS: Four/six participants in the 10-microL group, 1/6 in the 50-microL group and 2/6 in the 250-microL group developed parasitaemia. Geometric mean (GM) pre-patent periods were 13.9, 14.0 and 15.0 days, respectively. Six/six (100%) participants developed parasitaemia in the 25,000 and 75,000 PfSPZ IM and 3,200 PfSPZ DVI groups. GM pre-patent periods were 12.2, 11.4 and 11.4 days, respectively. Injection of PfSPZ Challenge was well tolerated and safe in all groups. CONCLUSIONS: IM injection of 75,000 PfSPZ and DVI injection of 3,200 PfSPZ resulted in infection rates and pre-patent periods comparable to the bite of five PfSPZ-infected mosquitoes. Remarkably, it required 23.4-fold more PfSPZ administered IM than DVI to achieve the same parasite kinetics. These results allow for translation of CHMI from research to routine use, and inoculation of PfSPZ by IM and DVI regimens. TRIAL REGISTRATION: ClinicalTrials.gov NCT01771848.