We compared the intracellular processing of the fusion (F) glycoproteins of an acute measles virus (MV) Nagahata strain and its relative Biken strain that caused subacute sclerosing panencephalitis (SSPE), Nagahata strain synthesizes a precursor F0 which acquires three asparagine (N)-linked oligosaccharide chains sequentially in 1 h. One oligosaccharide chain on the partially glycosylated F0 is less accessible to endo-beta-N-acetylglucosaminidase H (endo-H) but becomes accessible as the protein becomes fully glycosylated, suggesting a protein conformational change. Biken strain SSPE virus synthesizes a similarly glycosylated F0. However, one oligosaccharide chain on the Biken F0 remains less accessible to endo-H even after the protein is fully glycosylated. The Nagahata F0 is cleaved into the F1 and F2 subunits with a half life of 1 h. The Biken F0 is cleaved with a half life of 4 h. We cloned the F genes of Nagahata and Biken strains and showed by transfection that the defect causing delayed cleavage of F0 resides in the Biken F gene. Sequence analysis predicts a mutation in the cleavage recognition sequence, a truncated carboxyl-terminus, and multiple mutations in F1 of the Biken F protein. Expression of chimeric F genes showed the mutated cleavage recognition sequence and the carboxyl-terminal truncation do not delay cleavage of F0. Instead, delayed F0 cleavage is due to multiple mutations in the extracellular domain of F1, and four amino acid substitutions near the transmembrane region impair endo-H access to the oligosaccharide chain. These results provide detailed information on the normal maturation process of the F protein of MV and additional clues to the mechanisms of MV persistence in the CNS.