The natural mutations observed in pyruvate kinase (PK)-M2, a homotetramer isozyme, in this study correlated with the differential activity of the enzyme in a dominant negative manner in B-lymphoblastoid cells, established from two Bloom syndrome (BS) patients, BS1 and BS3 by 50 and 90%, respectively; and by 75% in the freshly obtained PHA stimulated lymphocytes of a BS patient diagnosed for the first time in India. A gene screen involving the critical domains of the PK-M gene in BS cells resulted in the observation of a missense mutation in BS1 and the BS patient and a frame shift mutation in BS3, in exon-10, coding for the intersubunit contact domain (ISCD) of the PK-M2 protein. Apart from these mutations, other variations in this region of the gene, both in normal and BS cells, did not affect the enzyme activity, since these were silent. Computer-based modeling studies of the PK-M2 protein with each mutation was suggestive of a changed interaction between two domains within a subunit in BS1, a gross structural change in BS3, and a changed interaction between two subunits of the tetramer in the BS patient. An absence of such mutations in other regions of the PK-M2 gene in normal subjects and in the randomly chosen unrelated genes in the DNA from BS cell lines and the cells from the BS patient, authenticated the presence of the observed mutations in Bloom syndrome cells. A correlation observed between the differential enzyme activity and the nature of mutation in the intersubunit contact domain (ISCD) region of the PK-M2 gene was interesting, and indicted how the site and the nature of mutation in a heterozygous state could influence the enzyme activity differentially and in a dominant negative manner. The importance of these mutations in Bloom syndrome cells, however, remains to be elucidated, and can only be conjectured.