Nucleosomal incorporation of specialized histone variants is an important mechanism to generate different functional chromatin states. Here, we describe the identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y. Their messenger RNAs are found in certain human cell lines, in addition to several normal and malignant human tissues. In keeping with their primate specificity, H3.X and H3.Y are detected in different brain regions. Transgenic H3.X and H3.Y proteins are stably incorporated into chromatin in a similar fashion to the known H3 variants. Importantly, we demonstrate biochemically and by mass spectrometry that endogenous H3.Y protein exists in vivo, and that stress stimuli, such as starvation and cellular density, increase the abundance of H3.Y-expressing cells. Global transcriptome analysis revealed that knockdown of H3.Y affects cell growth and leads to changes in the expression of many genes involved in cell cycle control. Thus, H3.Y is a novel histone variant involved in the regulation of cellular responses to outside stimuli.