HIV-1 genes are expressed through the complex splicing of a single mRNA precursor leading to three mRNA classes: unspliced, singly-spliced and multiply-spliced. Each class may include several mRNA species specifically encoding one or two HIV-1 proteins. Northern blotting and RT-PCR are the techniques currently used to analyse HIV-1 mRNA expression. Northern blotting allows quantitative detection of these three classes of viral RNA but does not discriminate between individual RNA species. RT-PCR allows discrimination between different species but does not provide a quantitative analysis. Here, we describe an application of an RNAse mapping assay which gives both quantitative and discriminative HIV-1 RNA detection. A radiolabeled probe overlapping the major splicing sites of HIV-1 used for the generation of HIV-1 mRNA subspecies was synthesized. This probe protects differential sizes of these species, allowing discrimination between them. We investigated the RNA expression pattern in high titer HIV-1 producing cells. The HIV-1-specific probe allowed the detection of multiply-spliced vpr, rev and nef mRNAs, singly-spliced env mRNA and unspliced genomic RNA. With its discriminative and quantitative properties, this application is particularly convenient for the investigation of HIV-1 mRNA expression during the course of HIV-1 infections.