This report describes the structure of soluble human stem cell factor isolated from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with stem cell factor (SCF) cDNA, which encodes a leader sequence plus 248 additional amino acids. The 248 amino acids include a hydrophobic transmembrane region at positions 190-212. The isolated material is glycosylated and three bands (apparent M(r) 28,000, M(r) 35,000, and M(r) 40,000) are evident by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After complete deglycosylation, the molecular weight by SDS-polyacrylamide gel electrophoresis is 18,000-19,000. Structural analyses of the intact SCF, the deglycosylated SCF, and a deglycosylated C-terminal peptide were performed by laser desorption, fast atom bombardment, or electrospray mass spectrometry. Pulse-labeling of cells with 35S-labeled Met and Cys resulted in cell-associated glycosylated SCF of M(r) 33,000-45,000 which was converted to M(r) 33,000 by in vitro treatment with glycosidases. During a chase with unlabeled Met and Cys, labeled SCF of M(r) 28,000, M(r) 35,000, and M(r) 40,000 appeared in the medium; it was converted to M(r) 18,000-19,000 by glycosidase treatment. SCF at the surface of the transfected CHO cells could be demonstrated by immunofluorescence. The data obtained indicate that the recombinant human stem cell factor, as isolated, represents proteolytically processed forms containing amino acids 1-165, derived from the initially synthesized membrane-bound form of 248 amino acids. Further characterization indicated that the M(r) 28,000 form is glycosylated at Asn120, the M(r) 35,000 form at Asn120 and Asn65, and the M(r) 40,000 form at Asn120, Asn93, and Asn65. Each form also contains O-linked carbohydrate. The N-linked glycosylation, particularly that at Asn93 and at Asn65, adversely affects in vitro biological activity and receptor binding.