Evidence is lacking on how best to decontaminate the hospital environment of Clostridium difficile. We compared sporulation levels in the UK epidemic C. difficile strain (P24), another clinical isolate (B31), and an environmental strain (E4) cultured in faecal emulsion containing subinhibitory concentrations of one of five hospital cleaning agents. The epidemic strain produced significantly more spores than the non-prevalent strains, and sporulation was further enhanced when this strain was cultured in faeces exposed to non-chlorine-based cleaning agents. The choice of cleaning agent can have a substantial effect on the persistence of C. difficile spores in the hospital environment.