When transferring the human multidrug resistance 1 (MDR1) cDNA, FMEV retroviral vectors mediate high-dose multidrug resistance and, thus, background-free selection in primary human hematopoietic progenitor cells. Here, we analyzed strategies for co-expression of a second gene from an FMEV:MDR1 vector. When linking the cDNAs with the internal ribosomal entry site (IRES) of poliovirus or retroviral splice signals, almost all multidrug-resistant hematopoietic colonies simultaneously coexpressed the 3' positioned second gene, neomycin-phosphotransferase (neoR). The IRES strategy allowed functional co-transfer of a 4.2-kb lacZ-neoR fusion gene, resulting in a total proviral genome size of 11 kb, corresponding to the packaging limit of retroviral vectors. Preselection based on multidrug resistance elevated the expression of the second gene in IRES constructs, but not in splice vectors. Moreover, three intriguing observations were made. First, up to 30% of cells preselected for functional transfer of the 3' positioned cDNA (neoR) showed infunctional MDR1; this occurred irrespective of the linking principle and was associated with instability of the MDR1 transcription unit. Second, the levels of multidrug resistance achieved with the co-expression vectors were moderately lower (15-30% reduced) than those mediated by the monocistronic counterpart. Third, transduction with FMEV:MDR1 co-expression vectors still resulted in high-dose cancer drug resistance and background-free selection of hematopoietic progenitor cells (including primary human CD34+ colony-forming units). Thus, for the first time, we describe MDR1 co-expression vectors that maintain their desired function in early and primary human hematopoietic cells. However, careful interpretation of the data reveals that further vector improvements are required to obtain clinically useful MDR1 co-expression vectors.