Ustilago maydis is a haploid basidiomycete with single genes for two distinct histone H3 variants. The solitary U1 gene codes for H3.1, predicted to be a replication-independent replacement histone. The U2 gene is paired with histone H4 and produces a putative replication-coupled H3.2 variant. These predictions were evaluated experimentally. U2 was confirmed to be highly expressed in the S phase and had reduced expression in hydroxyurea, and H3.2 protein was not incorporated into transcribed chromatin of stationary phase cells. Constitutive expression of U1 during growth produced ϳ25% of H3 as H3.1 protein, more highly acetylated than H3.2. The level of H3.1 increased when cell proliferation slowed, a hallmark of replacement histones. Half of new H3.1 incorporated into highly acetylated chromatin was lost with a half-life of 2.5 h, the fastest rate of replacement H3 turnover reported to date. This response reflects the characteristic incorporation of replacement H3 into transcribed chromatin, subject to continued nucleosome displacement and a loss of H3 as in animals and plants. Although the two H3 variants are functionally distinct, neither appears to be essential for vegetative growth. KO gene disruption transformants of the U1 and U2 loci produced viable cell lines. The structural and functional similarities of the Ustilago replication-coupled and replication-independent H3 variants with those in animals, in plants, and in ciliates are remarkable because these distinct histone H3 pairs of variants arose independently in each of these clades and in basidiomycetes.Core histones provide the packaging proteins for DNA in eukaryotic cells. During the S phase when genomic DNA is duplicated, replication-coupled expression of histone genes provides new protein to assemble newly replicated DNA. Histone chaperones assist in the creation of new nucleosomes to maintain a stable, compacted, repressed state of chromatin. Within this context, regulatory proteins modulate chromatin environments to facilitate access to the DNA for gene transcription. The components and processes that allow RNA polymerases to transcribe a chromatin template, such as epigenetic modifications of DNA and histones, are being identified and intensely studied. The processes of nucleosome displacement from DNA by transcribing RNA polymerases and of nucleosome reassembly from available histones remain poorly understood.In research going back decades, it was observed that the composition of nucleosomes across transcribed gene regions, identified in part by high levels of histone acetylation, changed over time. Replication-coupled (RC) 2 histone H3 variants like H3.2 in birds were replaced by histone H3.3, a constitutively expressed form of animal histone H3. This histone is now known as a replacement histone or as a replication-independent (RI) H3 variant (1, 2). Specialized chaperones such as HIRA and Daxx selectively bind these RI H3 proteins at a small region, residues 87-90, which is uniquely different between RI and RC forms (3, 4). In the S phase...