A cDNA encoding a bicistronic mRNA, E1E4L1, which was generated by double-splicing of the E1, E4 and L1 genes, of the type-6 human papillomavirus (HPV-6), was cloned. The E1E4 and L1 open reading frames (ORFs) in this cDNA were expressed in COS-1 or CV-1 cells as fusion proteins with Escherichia coli beta-galactosidase (beta Gal), and the products were analyzed by immunoprecipitation and enzyme assay. The results showed that the translational efficiency of the L1 ORF was about 9-15-fold less efficient than that of the E1E4 ORF. Substitution of the ATG of the E1E4 ORF with AAG increased translation of the L1 ORF about 30-fold. Lengthening of the intercistronic sequence to 31 bp, equivalent in length to the bicistronic HPV-1 mRNA, showed little translational effect relative to the wild-type 12-bp intercistronic sequence. (Carets [] represent splicing of RNA.)