Background: Coordinate enhancement of transgene transcription and translation would be a potent approach to significantly improve protein output in a broad array of viral vectors and nonviral expression systems. Many vector transgenes are complementary DNA (cDNA). The lack of splicing can significantly reduce the efficiency of their translation. Some retroviruses contain a 5′ terminal post-transcriptional control element (PCE) that facilitates translation of unspliced mRNA. Here we evaluated the potential for spleen necrosis virus PCE to stimulate protein production from HIV-1 based lentiviral vector by: 1) improving translation of the internal transgene transcript; and 2) functionally synergizing with a transcriptional enhancer to achieve coordinate increases in RNA synthesis and translation. Results: Derivatives of HIV-1 SIN self-inactivating lentiviral vector were created that contain PCE and cytomegalovirus immediate early enhancer (CMV IE). Results from transfected cells and four different transduced cell types indicate that: 1) PCE enhanced transgene protein synthesis; 2) transcription from the internal promoter is enhanced by CMV IE; 3) PCE and CMV IE functioned synergistically to significantly increase transgene protein yield; 4) the magnitude of translation enhancement by PCE was similar in transfected and transduced cells; 5) differences were observed in steady state level of PCE vector RNA in transfected and transduced cells; 6) the lower steady state was not attributable to reduced RNA stability, but to lower cytoplasmic accumulation in transduced cells. Conclusion: PCE is a useful tool to improve post-transcriptional expression of lentiviral vector transgene. Coordinate enhancement of transcription and translation is conferred by the combination of PCE with CMV IE transcriptional enhancer and increased protein yield up to 11 to 17-fold in transfected cells. The incorporation of the vector provirus into chromatin correlated with reduced cytoplasmic accumulation of PCE transgene RNA. We speculate that epigenetic modulation of promoter activity altered cotranscriptional recruitment of RNA processing factors and reduced the availability of fully processed transcript or the efficiency of export from the nucleus. Our results provide an example of the dynamic interplay between the transcription and post-transcription steps of gene expression and document that introduction of heterologous gene expression signals can yield disparate effects in transfected versus transduced cells.