Two regions of cauliflower mosaic virus DNA were designed as markers to study pathogen transmission in a child care home (CCH) and child care center (CCC) and in homes of CCC children. The DNA markers were stable for 1 month in the environment. The DNA markers were introduced into the environment through sensitized objects, and spread in the environment was traced by detection of the markers with polymerase chain reaction. The DNA markers spread rapidly in both the CCH and CCC after introduction and spread more rapidly in the toddler room than in the infant room of the CCC. Hand touching of contaminated areas was the major factor leading to spread of the markers. Hand washing and surface wiping decreased spread of the markers. The markers spread minimally from room to room in the CCC but were detected in the children's homes after introduction of markers in the CCC.