Abstract Cutaneous and mucosal carriage of Staphylococcus intermedius was determined in five Cavalier King Charles spaniel bitches, living in normal domestic environment, and their 18 puppies during the immediate prepartum period and after whelping, over a total of 8 weeks, using quantitative swab techniques. Within 8 h of birth S. intermedius was isolated from each puppy. The nose and mouth were the most frequently colonized mucosal sites (83 and 78%, respectively). At that time S. intermedius was isolated from the abdominal skin in 78% of the puppies. One day postpartum the mucosal populations increased significantly (P < 0.001) but fell 6 (P < 0.01) and 20 (P < 0.001) days later. Populations from abdominal skin reduced during the first and fourth weeks postpartum (P < 0.05 for both). This study indicates that in the domestic environment S. intermedius establishes itself soon after birth. The level of colonization by S. intermedius in the bitches seems to influence the colonization by pathogenic staphylococci in puppies.