Sporulation involves inducing scores of genes in a temporally and spatially coordinated cell development programme. Its initiation is controlled by an expanded two-component signal transduction system, termed a phosphorelay. The Spo0A response regulator is the master control element in the decision to sporulate, consisting of a receiver or phosphoacceptor domain and an effector or transcription activation domain having a high degree of sequence conservation. This study examined long-term Spo0A protein evolution for determining whether concerted evolution or purifying selection was the major factor in its evolution. The proportion of synonymous (pS) and non-synonymous nucleotide (pN) differences between Spo0A genes from 28 bacterial species was analysed. DIVERGE software was used for evaluating site-specific amino acids before phylogenetic divergence between Bacillus and Clostridium for detecting site-selective constrains operating on the Spo0A protein. Evolutionary Trace software was used for tracing the phylogenetic markers for this protein.