Tail lysozyme of bacteriophage T4, product of gene 5 (gp5), functions upon infection by locally digging a hole in the peptidoglycan layer, so that the tail tube, through which the phage DNA is injected, can penetrate to the inner membrane. It has been inferred from DNA sequence and expression of the tail lysozyme on a plasmid in Escherichia coli that the tail lysozyme is synthesized as a precursor of 62 K and is later cleaved to form a mature tail lysozyme of 42 K. Furthermore, gp5 has a region that is highly homologous to T4 lysozyme, gpe, that is the product of gene e and functions for 'lysis from within'. As an approach to elucidation of structure-function relationship of gp5, we determined mutational sites of gene 5 mutants that have heat sensitive virions, are temperature sensitive for growth, or require an amber suppressor. All the mutational sites were mapped in the region corresponding to the mature tail lysozyme. Among the ts mutants, 5ts1 was a pseudo-revertant of an amber mutant which bypasses gene e. It was mapped in the region which had a high homology to gpe, which is well known as T4 lysozyme. The other mutational sites will be also discussed in relation to the phenotypes of the mutants.