Legionella pneumophila serogroup 1 strains isolated from a cooling tower during the investigation of an outbreak of Legionnaires' disease were shown previously to be related closely or indistinguishable by hybridisation-based restriction fragment length polymorphism analysis. However, these strains could be differentiated into five different MAb subgroups by comparison of their reactivity patterns with a recognised panel of monoclonal antibodies (MAbs). Pulsed-field gel electrophoresis (PFGE) of genomic fragments obtained after cleavage with rare-cutting restriction endonucleases also differentiated these strains. Four different restriction patterns were obtained with SfiI, EagI and SmaI, three restriction patterns with NotI, ApaI and SacII, and two patterns with NaeI. Generally, the restriction patterns were related closely, differing in only one or two bands. The combined results of the restriction endonuclease digestions allowed the strains to be differentiated into groups that correlated to the MAb subgroups. Both PFGE patterns and MAb subgroups were found to be stable markers. The findings demonstrated that the MAb variability seen amongst the L. pneumophilia serogroup 1 strains from this cooling tower was not solely phenotypic.