Thirty-two related and 68 unrelated isolates of Clostridium difficile, isolated in different Italian hospitals since 1987, were analysed by PFGE and PCR-ribotyping to investigate their genetic relatedness. The isolates were classified into 28 groups by PFGE and 20 ribotypes by PCR-ribotyping. A single clone of C. difficile was recognised as the cause of three geographically and chronologically distant outbreaks. The correlation between PFGE and PCR-ribotyping results was good, with agreement for 77 (84%) of the 92 isolates typed by both methods. However, among sporadic isolates the discriminatory power of PFGE was more evident. Eight isolates that were untypable by PFGE could be analysed by PCR-ribotyping. The dendrograms generated showed that the genetic relatedness of the C. difficile isolates obtained by both techniques was comparable. The majority of the isolates in recent years appeared to be genetically unrelated to isolates from past infections. However, two clonal groups identified in all time periods had a common origin and this seems to indicate that they share some advantageous biological characteristics. The constant monitoring of C. difficile epidemiology will allow acquisition of further important data on this nosocomial pathogen.