The duration of feeding by larval Ixodes dammini was directly correlated with the proportion of larvae acquiring infection with the Lyme disease spirochete. The infection rate of ticks attached to hosts for 8 h (1.4%) was much lower than ticks feeding to repletion (87.5%). Infection was also detected in partially fed larvae dropping off dead animals. These partially fed larvae successfully reattached to rodents, fed to repletion, and molted to nymphs. Partially fed larvae did not transmit Borrelia burgdorferi when refeeding as larvae, but the resultant nymphs were capable of transmitting spirochetes. Infected host-seeking larvae collected in nature cannot be judged a priori to have acquired B. burgdorferi transovarially.