All three genes encoding histone H3 proteins were cloned and sequenced from Tetrahymena thermophila. Two of these genes encode a major H3 protein identical to that of T. pyriformis and 87% identical to the major H3 of vertebrates. The third gene encodes hv2, a quantitatively minor replication independent (replacement) variant. The sequence of hv2 is only 85% identical to the animal replacement variant H3.3 and is the most divergent H3 replacement variant described. Phylogenetic analysis of 73 H3 protein sequences suggests that hv2, H3.3, and the plant replacement variant H3.III evolved independently, and that H3.3 is not the ancestral H3 gene, as was previously suggested (Wells, D., Bains, W., and Kedes, L. 1986, J. Mol. Evol., 23: 224-241). These results suggest it is the replication independence and not the particular protein sequence that is important in the function of H3 replacement variants.