Colony stimulating factor-1 (CSF-1) is a homodimeric glycoprotein that humorally regulates the proliferation and differentiation of mononuclear phagocytic cells and locally regulates cells of the female reproductive tract. Alternative splicing of the human CSF-1 mRNA leads to alternative expression of the CSF-1 homodimer as a secreted glycoprotein or as a membrane-spanning molecule with cell surface biological activity. In the present study, analysis of immunoaffinity-purified CSF-1 from mouse L929 cell medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that CSF-1 is predominantly secreted as highly sulfated species of 375- and 250-kDa with a smaller amount of a 100-kDa species. Analysis by gel filtration in 4 M guanidine HCI buffer, indicated that, in contrast to the 100-kDa species, the highly sulfated species exhibit anomalously high molecular weights and self-association on SDS-PAGE similar to the dermatan sulfate proteoglycan, biglycan. The three predominant CSF-1 species were shown to be an 80-kDa homodimer, an 80-kDa/50-kDa heterodimer, and a 50-kDa homodimer. The 80-kDa subunit contained a single 18-kDa chondroitin sulfate chain that was absent from the 50-kDa subunit. Furthermore, treatment of the 80- and 50-kDa subunits, synthesized in the presence of tunicamycin, with chondroitinase ABC, neuraminidase, and endo-alpha-N-acetyl galactosaminidase reduced their apparent molecular masses to 60 and 25 kDa, respectively. These results are consistent with intracellular proteolytic cleavage of the 80-kDa chondroitin sulfate containing subunits from the membrane spanning CSF-1 precursor at a point carboxyl-terminal to the single consensus sequence for glycosaminoglycan addition and cleavage of the 50-kDa glycoprotein subunit at a position aminoterminal to this site. The predominance of the proteoglycan form of secreted CSF-1, which represents only 3-4% of the total trichloroacetic acid-precipitable counts released from 35SO4(2-)-labeled L cells, has important implications for regulation by this growth factor.