The proteins of nontypable and type b Haemophilus influenzae isolates were characterised using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Coomassie Brilliant Blue R-250 was used for protein detection. Two hundred and twenty eight proteins were resolved from whole cell lysates prepared from a standard nontypable H. influenzae strain (designated HI–64443) when isoelectric focusing was used for the first-dimensional separation of 2-D PAGE. When nonequilibrium pH gel electrophoresis (NEPHGE) was used to separate basic proteins in the first dimension, 50 proteins were detected for HI–64443; 20 of the basic proteins detected were considered to be unique for this separation protocol. The apparent molecular weights and isoelectric points were determined for 82 of the proteins resolved for HI–64443. The variation of the proteins from the standard bacterial strain (HI–64443) was determined for nontypable H. influenzae isolates. On the basis of their electrophoretic mobilities, 17.5% of the proteins of HI–64443 were shared by four other non-typable H. influenzae strains analysed. These data identified both conserved and variable proteins among the nontypable H. influenzae isolates analysed. The results obtained indicated that 2-D PAGE was able to discriminate nontypable H. influenzae into population clones identified by other procedures. The 2-D protein profiles obtained for type b H. influenzae strains were similar to those obtained for nontypable H. influenzae strains. The extent of the protein variation observed between type b and nontypable H. influenzae strain was similar to that observed among nontypable strains alone. These data are discussed in relation to the application of 2-D PAGE as a tool for studies on bacterial epidemiology and for the analysis of the genome structure and gene expression of Haemophilus influenzae.