The initial sites of vaccinia virus replication in the placenta are in cells lining maternal blood spaces in the trophospongium. Females were sham infected or infected intravenously with 2 × 106 PFU of VVWR on the 12th day of pregnancy. Placentas were harvested 4 days later, embedded in paraffin, subjected to in situ hybridization with a vaccinia virus-specific probe for the viral early gene C11R, and counterstained with hematoxylin and eosin as described in Materials and Methods. (A) Representative uninfected placenta (magnification, ×5). (B) Infected placenta showing viral mRNA in cells lining maternal blood spaces within the trophospongium layer of the placenta (magnification, ×5). (C) Higher magnification (×20) of the area within the box outlined in panel B. Arrows indicate infected cells.