C. difficile is a major cause of nosocomial diarrhoea. The toxins produced by C. difficile are responsible for the characteristic pathology observed in CDI, but several surface-associated proteins of C. difficile are also recognised by the immune system and could modulate the immune response in infection. The aim of this study was to assess the induction of cytokines in a macrophage cell line in response to different antigens prepared from five C. difficile strains: the hypervirulent ribotype 027, ribotypes 001 and 106 and reference strains VPI 10463 and 630 (ribotype 012). PMA activated THP-1 cells were challenged with surface-layer proteins, flagella, heat-shock proteins induced at 42°C and 60°C and culture supernatants of the five C. difficile strains. The production of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8 and IL-12p70 was observed in response to the surface-associated proteins and high levels of TNF-α, IL-1β and IL-8 were detected in response to challenge with culture supernatants. The immune response triggered by the surface-associated proteins was independent of the strain from which the antigens were derived, suggesting that these proteins might not be related to the varying virulence of the hypervirulent ribotype 027 or ribotypes 001 and 106. There was no inter-strain difference observed in response to the culture supernatants of the tested C. difficile strains but this was perhaps due to toxicity induced in the macrophages by large amounts of toxin A and toxin B. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.