The expression of two or more genes from a single viral vector has been widely used to label or select for cells containing the transgenic element. Identification of the foot-and-mouth disease virus (FMDV) 2A cleavage peptide as a polycistronic linker capable of producing equivalent levels of transgene expression has greatly improved this approach in the field of gene therapy. However, as a consequence of 2A posttranslational cleavage the upstream protein is left with a residual 19 amino acids from the 2A sequence on its carboxy terminus, and the downstream protein is left with an additional 2 to 5 amino acids on its amino terminus. Here we have assessed the functional consequences of the FMDV 2A cleavage motif on two secreted proteins (interleukin [IL]-2 and transforming growth factor [TGF]-β) when expressed from a retroviral bicistronic vector. Whereas IL-2 expression and function were found to be unaffected by the 2A motif in either orientation, functional expression of secreted TGF-β was significantly abrogated when the transgene was expressed upstream of the 2A sequence. We believe this is a consequence of aberrant cleavage and intracellular trafficking of the TGF-β polyprotein. These results highlight that to achieve functional expression of secreted proteins consideration must be taken of the transgenic protein's posttranslational modification and trafficking when using 2A-based bicistronic cassettes.