Analysis of the restriction enzyme digests of total genomic DNAs from a broad spectrum of human cell lines and from individuals with different genetic backgrounds, by hybridization with a series of cloned human histone sequences, indicated restriction site polymorphisms (RSPs) for two adjacent human histone genes which reside on chromosome 1. In most cell lines and individuals examined we observed a single 2.05 kb H4 histone HindIII fragment and a 7.0 kb H3 histone HindIII fragment. In contrast, the polymorphisms were manifested as a 2.15 kb H4 HindIII fragment and a 9.1 kb H3 HindIII fragment. From population studies, we were able to show that there is no linkage disequilibrium between these two polymorphic restriction sites. Nor was there any apparent correlation between the presence of the H3/H4 histone polymorphisms and maintenance of the transformed karyotype, passage in culture, transformation or tumor progression. These chromosome 1 H3 and H4 histone gene polymorphisms are common in the American Black population and, in our survey of individuals, were not found in the American Caucasian population. Among the American Blacks studied, the frequency of the H3 HindIII(-) allele is 43% and of the H4 HindIII(-) allele 30%. In limited family studies, we were unable to detect recombination between these two physically linked alleles.