Rat alpha-fetoprotein (RAFP) shows two discrete electrophoretic variants, slow and fast, with molecular weights of 72,500 and 69,500, respectively. To elucidate the structural basis of this heterogeneity, we developed the expression system of RAFP cDNA and its mutants with the use of cultured mouse fibroblasts and a retrovirus vector. This produced recombinant wild-type and three mutant proteins and has several advantages over the use of Escherichia coli or Saccharomyces cerevisiae. The mutants were designed to lack either one or both of the two possible glycosylation sites on RAFP. Electrophoretic analyses relating to the glycosylation states of the recombinant proteins as well as of natural RAFP produced by rat cells before and after digestion with glycopeptidase F indicated that the slow variant has carbohydrate units located at Asn-93 and Asn-229 and the fast one has one at Asn-229.