The duration of Borrelia burgdorferi infectivity in white-footed mice (Peromyscus leucopus) experimentally inoculated or infested with infected Ixodes scapularis nymphs was evaluated. Infectivity was assessed by infesting these mice with unfed I. scapularis larvae at 7, 21, 35 and 49 days post-inoculation (DPI) or post-infestation (PI). At 7 DPI, B. burgdorferi was transmitted from 18 of 24 syringe-inoculated mice and all three tick-infected mice to I. scapularis larvae which fed upon them. However, at 21, 35 and 49 DPI, significantly fewer mice were infective. Borrelia burgdorferi was isolated from tissues of 14 of 22 syringe-inoculated mice about 56 DPI, and from all three tick-infected mice. However, the level of agreement between xeno-diagnosis and bacterial culture was no greater than would be expected by chance alone. We also determined if B. burgdorferi infectivity of mice varied in relation to periods of tick feeding in the field. White-footed mice were trapped during April, July and August 1993 from two habitats on Long Point peninsula (Ontario, Canada), where B. burgdorferi is endemic. Mice from each habitat were infested with laboratory-reared I. scapularis larvae. Ticks from each mouse were subsequently examined by immunofluorescent assay for B. burgdorferi infection and mice were cultured for B. burgdorferi. None of 3577 I. scapularis larvae fed on 62 mice captured within the cottonwood dune habitat were infected with B. burgdorferi, although it was isolated from six of these mice. Within the maple forest habitat, 0/24, 8/21 (38%) and 1/21 (5%) mice transmitted B. burgdorferi to I. scapularis larvae during April, July and August, respectively. Most mice from the maple forest with B. burgdorferi-positive tissues (14/21) were collected during July, although the level of agreement between xenodiagnosis and tissue culture was poor. Because B. burgdorferi infectivity in mice appears to be of short duration, overwintered I. scapularis larvae and nymphs may have to feed upon infected hosts at the same time of year in order for a cycle of B. burgdorferi infection to be maintained on Long Point. Infected I. scapularis nymphs, rather than persistently infected vertebrate hosts, likely serve as the overwintering “reservoir” for B. burgdorferi on Long Point.