The effects of serum fractions on the growth kinetics and colony formation of mononuclear phagocytes derived from mouse bone marrow, blood, and peritoneal cavity were investigated. Peritoneal exudate macrophages and blood monocytes required a factor(s) found to reside in the nondialyzable serum fraction (molecular weight greater than 12,000) to survive, a small molecular weight (less than 307) factor(s) with growth-stimulatory activity (GSA) contained in the dialyzable serum fraction, and the macrophage growth factor (MGF) for proliferation and colony formation. Fetuin, a major protein of fetal serum, was able to substitute the nondialyzable serum fraction. Macrophages cultured in medium containing MGF and the nondialyzable serum fraction for 6 days could be restored to full growth following the addition of the dialyzable serum fraction. In contrast, bone marrow mononuclear phagocytes cultured in the absence of the dialyzable serum fraction were capable of proliferating, though at a slower rate, and forming colonies. In addition, neither insulin nor hydrocortisone was capable of replacing the serum-dialyzable GSA nor able to enhance colony formation.