The abundant human papillomavirus type 11 (HPV 11) E1E4,E5 transcript potentially encodes the E1E4,E5a and E5b proteins. It is not known if either of the E5 proteins are expressed from this transcript. For HPV 16, E5 is a single open reading frame (ORF), and the E5 protein is expressed from an unspliced E2,E5 transcript but not from the spliced E1E4,E5 transcript. This study was undertaken to determine if the HPV 11 E5a protein is expressed from the E1E4,E5 transcript. To detect E5a expression in eukaryotic cells, the green fluorescent protein (GFP) gene was fused to the 3' end of the E5a gene in the pEGFP-N1 vector. Several recombinant plasmid constructs were made to determine if E5a translation is influenced by upstream sequences present in the E1E4,E5 transcript. COS-7 cells were transfected with each construct, and flow cytometry was performed after 24 h of growth. The amount of E5a-GFP expressed from each construct was determined by the mean fluorescence of 2,000 transfected cells. Although the E5a-GFP fusion was expressed by all but one construct, the quantity of expressed E5a-GFP varied considerably. The most abundant expression was detected in cells transfected with the E1E4,E5a construct that lacked the 5' noncoding sequence between nucleotides (nts) 714 and 831 that is present in the authentic transcript. Other constructs expressed E5a-GFP in variable amounts, suggesting that sequences between nt 714 and the start of the E5a ORF affect expression of the E5a protein. An E2,E5a construct was made to compare the HPV 11 E5a expression to that of HPV 16. In contrast to HPV 16, no E5a-GFP was expressed from the HPV 11 E2,E5a construct. E1wedgeE4 protein was detected by immunofluorescence in COS-7 cells transfected with a construct that expressed E1E4 as a T7-epitope-tagged protein, and E5a as a GFP fusion. We conclude that the abundant HPV 11 E1E4,E5 transcript is a functional message that can support both E1E4 and E5a expression in eukaryotic cells.