Human immunodeficiency virus type 1 (HIV-1) transcripts have three fates: to serve as genomic RNAs, unspliced mRNAs, or spliced subgenomic mRNAs. Recent structural studies have shown that sequences near the 5' end of HIV-1 RNA can adopt at least two alternate three-dimensional conformations, and that these structures dictate genome versus unspliced mRNA fates. HIV-1's use of alternate transcription start sites (TSS) can influence which RNA conformer is generated, and this choice, in turn, dictates the fate of the unspliced RNA. The structural context of HIV-1's major 5' splice site differs in these two RNA conformers, suggesting that the conformers may differ in their ability to support HIV-1 splicing events. Here, we tested the hypothesis that TSS that shift the RNA monomer/dimer structural equilibrium away from the splice site sequestering dimer-competent fold would favor splicing. Consistent with this hypothesis, the results showed that the 5' ends of spliced HIV-1 RNAs were enriched in 3G structures and depleted of 1G RNAs relative to the total intracellular RNA population. These findings expand the functional significance of HIV-1 RNA structural dynamics by demonstrating roles for RNA structure in defining all three classes of HIV-1 RNAs, and suggest that HIV-1 TSS choice initiates a cascade of molecular events that dictate the fates of nascent HIV-1 RNAs.