Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90% similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.