Histones through a complex repertoire of non-allelic variants and their post-translational modifications regulate gene expression. Though alterations in histone-modifying enzymes and post-translational modifications of histones have been studied in cancer, expression of histone variants has not been clearly associated with dedifferentiation and malignant transformation of hepatocyte in vivo. In the present work, the pattern of variants of histones was investigated during N-nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis. Our studies show for the first time in vivo overexpression of a major histone H2A variant H2A.1 and a decrease in H2A.2 at protein and mRNA levels by sodium dodecyl sulfate-Acetic acid-Urea-Triton (SDS-AUT) two-dimensional gel electrophoresis followed by matrix-assisted-laser desorption/ionization time-of-flight (TOF)/TOF mass spectrometry and reverse transcriptase-polymerase chain reaction analysis during sequential development of hepatocellular carcinoma (HCC). H2A.1 and H2A.2 are highly homologous, replication-dependent, non-allelic variants of histone H2A differing at only three amino acid positions. Our results of increase in proliferating cell nuclear antigen expression indicate that with increase in replicating population of transformed cells in HCC, H2A.1 expression increases, suggesting association of H2A.1 overexpression with hyper-proliferation of hepatocytes during cellular dedifferentiation and progressive transformation of normal liver to preneoplastic and neoplastic stages of HCC.