HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5′ splice site upstream of the env open reading frame. To determine the role of this splice site in the 5′-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5′ splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5′ splice site, SD4, and the free 5′ end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5′ splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions −3 to +8 of the 5′ splice site and all 11 nt constituting the single-stranded 5′ end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4.