The incidence of Clostridium difficile-associated infections is increasing, but it remains to be defined whether any change in the seroprevalence of C. difficile has also occurred. In a population-based study of the general adult population, 734 subjects, aged 15–69 years, were examined on two occasions 8 years apart (1990 and 1998) for the presence of antibodies against C. difficile in serum. The overall seroprevalence of C. difficile increased significantly from 19% in 1990 to 27% in 1998 (P<0·0001). The seroprevalence increased with increasing age in both 1990 and 1998, but the increase was about four times higher in 1998 than in 1990. In conclusion, the observed increase in seroprevalence suggests a higher exposure to C. difficile in the general Danish adult population.