Staphlylococcus aureus is the primary pathogen associated with osteomyelitis, an acute and recurrent bone disease. Internalization of S. aureus by cultured embryonic chick calvarial osteoblasts has been observed. The purpose of this study was to demonstrate that internalization of bacteria by embryonic chick calvarial and tibial osteoblasts occurs in vivo. In initial experiments, 10(8) colony forming units (cfu) of S. aureus, strain UAMS-1 or Cowan 1, were injected subcutaneously under the scalp skin of 17 day chick embryos. After 45 min, calvariae were harvested and processed for transmission electron microscopy (TEM). In subsequent experiments, 10(9) cfu of UAMS-1 were injected into the allantoic sac of 17 day chick embryos via a small opening in the egg shell. After 48 h, calvariae and tibiae were harvested for TEM. S. aureus cells were found in approximately 14% of the calvarial osteoblasts after subcutaneous injection and in 11% of calvarial and tibial osteoblasts following intraallantoic injection. Endosomes were observed in some cells, but most bacteria internalized appeared to be free in the cytoplasm. Osteoblasts with as few as five bacteria had a greater loss of cytoplasmic integrity and a more heterochromatic nucleus than osteoblasts with fewer bacteria or than uninfected osteoblasts. S. aureus cells in calvariae and tibiae were also observed in the cytoplasm of approximately 4% of the osteocytes in mineralized bone matrix. Thus, internalization of S. aureus by osteoblasts in vivo augments the previous observation in vitro. This study has also shown that osteoblasts with few bacteria continue differentiating into osteocytes. Results of these experiments support the hypothesis that internalization of S. aureus by osteoblasts may play a role in the etiology of osteomyelitis.