We investigated how long an infected I. scapularis nymph must feed on a susceptible host for successful transmission of A. phagocytophilum. Individual nymphs were placed upon BALB/c mice and allowed to attach for various intervals between 4 and 78 hours. The success of transmission was assessed by PCR on mouse blood at 7, 14 and 21 days postinfestation, and IFA at 21 days postinfestation. Only 17-33% of infected ticks allowed to remain attached for up to 16 hours transmitted A. phagocytophilum to susceptible animals, versus 60% of those allowed to feed for 24 hours. The transmission success reached 93% in ticks attached for 78 hours. Interestingly, none of the PCR-positive mice that were exposed to infected ticks for 4 to 8 hours and only a half of those PCR -positive mice exposed for 12 to 20 hours seroconverted within 3 weeks postinfestation. On the other hand, all mice that turned PCR-positive after being infested for at least 24 hours seroconverted. This suggests that although some ticks feeding for less than 24 hours do inject a small amount of A. phagocytophilum, this amount is insufficient for stimulating humoral immunity and perhaps for establishing disseminated infection in BALB/c mice. Although A. phagocytophilum is present in salivary glands of unfed I. scapularis nymphs, the amount of A. phagocytophilum initially contained in saliva appears insufficient to cause infection in a host; stimulation of replication of the agent for 12-24 hours in a feeding tick is required before a host mouse can be consistently infected.