The presence of outer surface protein A (OspA) as a marker for Borrelia burgdorferi in Ixodes scapularis ticks was monitored with an OspA antigen-capture ELISA. The OspA ELISA, with a sensitivity of 30 spirochetes (8 fg), was not affected by the presence of either recently blood-fed or flat tick homogenates. Median spirochete equivalent levels as high as 16,000 in larvae, 55,000 in attached nymphs, and 10,000 in unfed adults were observed. Estimates of OspA doubling times ranged from a maximum of 140 h in larvae to as short as 17.5 h during nymphal attachment to the host. Spirochete equivalents in nymphs fell by 3 x 10(4) spirochete equivalents (54% of total OspA) in the last 12 h of attachment to mice but rose after detachment. Each OspA-positive I. scapularis nymph, regardless of spirochete equivalent density or length of attachment, successfully transmitted B. burgdorferi to a mouse.