Abstract Polynucleotide phosphorylase (PNPase) is a polynucleotide nucleotidyl transferase (E. C. 22.214.171.124) that is involved in mRNA degradation in prokaryotes. PNPase structure analysis has been performed in Streptomyces antibioticus; this revealed the presence of five domains: two ribonuclease PH (RPH)-like (pnp1 and pnp2), one alpha helical, one KH, and one S1 domains. The trimeric nature of this enzyme was also confirmed. In this work, we have investigated conserved domains or subdomains in bacterial PNPases (55), for this structure-based sequence homology analysis between predicted amino acid sequences from bacterial PNPases and that of S. antibioticus was performed. Our findings indicate that while pnp2 (% similarity average S ¯ = 84/% identity average I ¯ = 22), KH ( S ¯ = 74.3%/ I ¯ = 5.3%), S1 ( S ¯ = 71.3%/ I ¯ = 1.2%); and pnp1 ( S ¯ = 52.8%/ I ¯ = 0.3%) domain; structure and sequence are well conserved among different bacteria, alpha helical domain ( S ¯ = 39.5%/ I ¯ = 0) although conservation of the structure is somewhat maintained, the sequence is not conserved at all. Implications of such findings in PNPase activity will be discussed.