FP21 is a glycoprotein within the cytosolic compartment of Dictyostelium which carries an unusual carbohydrate modification(s) including the sugars fucose, galactose and N-acetylglucosamine. The soluble pool of FP21 from crude extracts resolves chromatographically into two fractions that differ in their glycosylation. Previous gene-mapping studies indicating the existence of two loci suggested that the FP21 fractions might be encoded by different genes. To address this issue, the two genes were cloned and sequenced, leading to the prediction that the protein products would differ by only a single amino acid, Ser or Ala, at codon 39. Protein sequence data on CNBr fragments of purified FP21 showed that both gene products are found in both fractions of the soluble pool. After further purification, the two fractions were no longer chromatographically resolvable, and there was no evidence for charge heterogeneity as determined by 2-D gel electrophoresis of whole cells. Thus, the initial separation of the different soluble subpopulations of this protein appears to be due to distinct molecular complexes, possibly related to differential glycosylation, and is not the result of the genetically-encoded amino acid polymorphism.