Multigene expression vectors are commonly utilized whereby the (over)expression of two or more genes is desired simultaneously and often at supposedly equivalent levels. We have characterized dual-gene and pEE14.4 RlucMFluc expression plasmids in which the second hCMV-MIE promoter is replaced with a c-myc IRES to enable one-step coordinated expression of multiple genes in eukaryotic cells. The efficacy of these plasmids has been tested in Chinese hamster ovary (CHO) cells using renilla and firefly luciferase reporter genes, with the reporter gene in either position 1 or 2 from the 5' to 3' direction, respectively. The dual-gene constructs gave enhanced second position gene expression levels compared to the first gene during transient transfection experiments (4- to 50-fold increase 24 h post-transfection). The pEE14.4 RlucMFluc plasmid gave enhanced first cistron expression compared to the second cistron (similar to 19-fold increase 24 h post-transfection). More equivalent transient expression levels were obtained by undertaking co-transfection of the appropriate single gene plasmids. Reporter gene mRNA levels in the transfected cells were also evaluated by qRT-PCR and compared to the observed protein expression levels. Analysis of the resulting data showed that transcriptional limitation of the first cistron in the dual-gene expression system and not translational limitation was the major limiting factor. Taken together these data suggest that relative protein expression levels expected from heterologous gene products in a multigene vector cannot be predicted on copy number alone and it is important to characterize multigene or oligocistronic systems prior to use.