The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s) H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1), from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol) II. Most interestingly, many of these interactions with H2A.B.3 (Sf3b155, Spt6, DDX39A and RNA Pol II) were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly in vitro and in vivo, and associates with mRNA at intron—exon boundaries. This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors (Sf3b155, Spt6, DDX39A and RNA Pol II). Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS), but also at the beginning of the gene body (but being excluded from the +1 nucleosome) compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z. The substitution of core histones with their non-allelic variant forms plays a particular important role in regulating chromatin function because they can directly alter the structure of chromatin, and provide new protein interaction interfaces for the recruitment of proteins involved in gene expression. Despite being discovered over a decade ago, the function of H2A.B, a variant of the H2A class, in its proper physiological context (being expressed in the testis and the brain) is unknown. We provide strong evidence that H2A.B has a role in the processing of RNA. It is found in the gene body of an active gene, directly interacts with RNA polymerase II and splicing factors and is located in the nucleus at distinct regions enriched with RNA processing factors (splicing speckles). Most significantly, we show that H2A.B can directly bind to RNA both in vitro and in germ cells. Therefore, H2A.B has the novel ability to bind to both RNA and DNA (as well as proteins) thus directly linking chromatin structure with the function of RNA. Taken together, this suggests that a special mechanism of splicing may operate in the testis and brain.