In the course of studies on HIV-1 RNA structure, we determined that the main 5' end of viral RNA from virions and virus producer cells corresponds to G456 in the proviral DNA sequence, one or two nucleotides down-stream from the reported ends that correspond to G454 and G455. We mapped 5' ends using the highly accurate RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) method. The reactivity of the 5' ends indicates that they are mainly capped, although the presence of some uncapped (5'-triphosphorylated) RNA cannot formally be excluded. When we used a 5' mapping method susceptible to incorporating a cytosine at the 3' end of cDNA first strands, at a position templated by the 7-methylguanosine cap, 50% of clones derived from virion RNA had incorporated the additional cytosine. Reassignment of the 5' end has consequences for the design of short RNAs used to study HIV-1 RNA structural dynamics.