The major structural proteins of 13 temperate and one virulent Listeria typing bacteriophages were analysed and compared using isoelectric focusing in immobilized pH gradients (IPG), ultrathin-layer two-dimensional electrophoresis, amino acid analysis and N-terminal amino acid sequences of selected proteins. Isoelectric points for major capsid and tail proteins of the 12 members of the siphoviridae family included in this study ranged from 4.70 to 5.92, whereas one of the two myoviridae investigated (B054) showed structural proteins in the 6.1 to 6.3 range. In comparison to protein profiles from one-dimensional SDS gels, the IPG technique gave better resolution and improved discrimination of phage proteins. Combination of this technique and SDS gel electrophoresis made it possible to correlate M(r) and isoelectric points of major structural proteins. Tail polypeptides of all siphoviridae are smaller and, with one exception, more acidic than their corresponding capsid counterparts. We also determined the amino acid composition of capsid and tail proteins. When compared with an average protein, they were found to be fairly rich in acidic and short-chain hydrophobic amino acids, as well as in lysine. In addition, the N-terminal amino acid sequences of major capsid and tail proteins of four representative listeriaphages were compared. The base composition of listeriaphage DNAs was between 37% and 39% G + C, reflecting that of their bacterial hosts. Each phage had a distinct restriction endonuclease pattern, and genome sizes ranged from 35 to 116 kb. DNA-DNA hybridization permitted the identification of five DNA homology groups. The two myoviruses studied (A511 and B054) showed no DNA homology to other phages, confirming their unique nature. The 12 siphoviruses were classified into three DNA homology groups with little cross-homology. Furthermore, phage A006 was found to share little DNA homology with the other investigated members of species 2671. Therefore, a new species (A006) is proposed. With respect to phage classification and taxonomy, a good correlation between the various approaches was observed, mostly corresponding to particle morphology.