The cDNA sequence of eukaryotic translation initiation factor eIF4E was derived from a Spodoptera frugiperda cDNA library. Eight tryptophan residues, typical for eIF4E, are strictly conserved in the encoded 210 amino acid protein. A polyclonal antiserum detected a 26 kDa protein in lepidopteran cell lines, but not in dipteran cells. Sf21 cells have a single eIF4E gene copy, which is transcribed into a 1500 nt transcript. Infection with AcMNPV resulted in a decrease in eIF4E mRNA starting between 12 and 24 h postinfection (p.i.), while reduced eIF4E protein levels were observed at 48 h p.i. Two forms of eIF4E were recognized that differed in their iso-electric point, of which the relative abundance did not change during infection. Mutagenesis experiments using recombinant baculoviruses revealed that the variation in mobility between these two forms did not result from a difference in the phosphorylation state of Ser-202, the serine residue that corresponds with the eIF4E phosphorylation site in mammalian eIF4E.