Listeria monocytogenes causes foodborne outbreaks that lead to infection in human and other mammalian fetuses. To elucidate the molecular and cellular mechanisms involved in transplacental transmission, we characterized placental-fetal infection in pregnant guinea pigs inoculated with wild-type (wt) or mutant L. monocytogenes strains. The wt strain increased in number in the placenta by >1000-fold during the first 24 h after inoculation--an increase that was unparalleled in other maternal organs. The ActA- mutant, which is impaired in cell-to-cell spread and attenuated in maternal organs, increased in the placenta by a similar amount, although, in fetal infection, the number of ActA- mutant bacteria was 100-fold lower, compared with that of the wt strain. Furthermore, a mutant impaired in vacuolar escape was rapidly eliminated from maternal organs but persisted in the placenta. We concluded that cell-to-cell spread facilitates maternal-to-fetal transmission. Furthermore, the placenta provides a protective niche for growth of L. monocytogenes.