Foamy viruses (FV) comprise a subfamily of retroviruses. Orthoretroviruses, such as human immunodeficiency virus type 1, synthesize Gag and Pol from unspliced genomic RNA. However, FV Pol is expressed from a spliced mRNA independently of Gag. FV pol splicing uses a 3' splice site located at the 3' end of gag, resulting in a shared exon between gag and pol. Previously, our laboratory showed that C-terminal Gag premature termination codon (PTC) mutations in the 3' shared exon led to greatly decreased levels of Pol protein (C. R. Stenbak and M. L. Linial, J. Virol. 78:9423-9430, 2004). To further characterize these mutants, we quantitated the levels of unspliced gag and spliced pol mRNAs using a real-time PCR assay. In some of the PTC mutants, the levels of spliced pol mRNA were reduced as much as 30-fold, whereas levels of unspliced gag RNA were not affected. Substitutions of a missense codon in place of a PTC restored normal levels of spliced pol mRNA. Disrupting Upf proteins involved in nonsense-mediated mRNA decay (NMD) did not affect Pol protein expression. Introduction of an exonic splicing enhancer downstream of the PTC mutation restored pol splicing to the wild-type level. Taken together, our results show that the PTC mutation itself is responsible for decreased levels of pol mRNA but that mechanisms other than NMD might be involved in downregulating Pol expression. The results also suggest that normal pol splicing utilizes a suboptimal splice site seen for other spliced mRNAs in most retroviruses, in that introduced exonic enhancer elements can increase splicing efficiency.