Propagation of A. phagocytophilum in the natural transmission cycle depends on the ability of uninfected ticks to acquire the pathogen from a host and to transmit it through the moult to the next life-stage (transstadially). We studied the dynamics and efficiency of A. phagocytophilum acquisition and transstadial transmission by I. scapularis larvae. Uninfected larvae were placed upon infected mice at one and two weeks post-infection. Larvae feeding for 24, 48, and 72 hours, fully-engorged larvae, ticks digesting blood-meal for 28 d post-engorgement, as well as freshly-moulted nymphs (49 d post-engorgement) were tested by quantitative PCR. Prevalence of A. phagocytophilum in ticks fed at 1 wk post-infection was significantly higher than in those fed at 2 wks. However, the average load of the pathogen in the infected ticks was similar between the two infestations. The resulting amount of A. phagocytophilum in freshly moulted nymphs was 115,000-122,000 per infected tick. The estimated efficiency of transstadial transmission of A. phagocytophilum by I. scapularis larvae is 80-100%.