We studied expression of HPV 58 long and short L1 proteins in primary mouse keratinocyte (KC) cultures by transient transfection of the L1 expression constructs. Following transient transfection, long and short L1 open reading frames (ORFs) were transcribed continuously for 9 days; however, no significant difference was detected between the long and short L1 mRNA levels measured by quantitative RT-PCR. Western blot analysis showed that both long and short L1 proteins were continuously detected in L1-transfected KCs for 9 days post-transfection and the significantly increased signals of the L1 proteins over time were associated with KC differentiation. Moreover, L1 protein was more abundant in KCs transfected with the short L1 ORF than the long L1 ORF. In vitro translation of the L1 mRNAs indicated further that the short L1 mRNA had significantly higher translation efficiency than the long L1 mRNA in cell-free lysate system. The L1 proteins expressed from the two L1 mRNAs in KCs were similarly stable. Thus, approximate 40% lower level of expression of the L1 protein in KCs transfected with the long L1 ORF was probably due to a stem-loop structure with high ΔG value downstream the first AUG codon in its mRNA secondary structure. This stem-loop structure might prevent efficient binding of the ribosome to mRNA and therefore reduced translation.