Staphylococcus aureus forms giant colonies on soft-agar surfaces, which is called colony-spreading. In the present study, we searched for host factors that influence S. aureus colony-spreading activity. The addition of calf serum, porcine serum, or silkworm hemolymph to soft-agar medium stimulated S. aureus colony-spreading activity. Gel filtration column chromatography of calf serum produced a high molecular weight fraction and a low molecular weight fraction, both of which exhibited colony-spreading stimulatory activity. In the low molecular weight fraction, we identified the stimulatory factor as bovine serum albumin. The stimulatory fraction in the high molecular weight fraction was identified as high-density lipoprotein (HDL) particles. Delipidation of HDL abolished the stimulatory activity of HDL. Phosphatidylcholine, which is the major lipid component in HDL particles, stimulated the colony-spreading activity. Other phosphatidylcholine-containing lipoprotein particles, low-density lipoprotein and very low-density lipoprotein, also showed colony-spreading stimulatory activity. These findings suggest that S. aureus colony-spreading activity is stimulated by albumin and lipoprotein particles in mammalian serum.