Retroviral gene expression requires nuclear export and translation of incompletely spliced RNA. In the case of human immunodeficiency virus (HIV), this is facilitated by the viral Rev protein binding to its cognate RNA response element (RRE), while other retroviruses contain constitutive transport elements (CTE) binding to cellular factors. These CTE can substitute for the HIV-1 Rev/RRE system, albeit with reduced efficiency. Here, we show that multimeric copies of the CTE restore HIV-1 protein expression to levels comparable to or higher than Rev/RRE in various cell lines from different species. We suggest that multimerization of export factors is important for CTE function, as reported for Rev. CTE function was not affected when the element was displaced from its natural position close to the poly(A) signal, while insertion of an intron into the 3'-untranslated region (3'-UTR) severely reduced CTE activity. In this case, cytoplasmic RNA degradation was observed, which may be mediated by nonsense-mediated RNA decay. In contrast, Rev-dependent gene expression was insensitive to an intron in the 3'-UTR. Finally, we show that the putative CTE-binding protein RNA helicase A is not specifically translocated into the cytoplasm upon overexpression of CTE-containing RNA.