Pulsed-field gel electrophoresis (PFGE) analysis of restriction pattern polymorphism was applied to type Clostridium difficile isolated from neonates hospitalized in a neonatal intensive care unit, and the results were compared with those of immunoblot analysis. C. difficile was isolated from fecal specimens of 41 (61%) of 67 neonates during a 5-month investigation. All of these neonates were asymptomatic. Fifty-five C. difficile isolates from 32 patients were analyzed by PFGE after digestion with SmaI and SacII endonucleases and by immunoblotting with 10 different antisera. Fifty-three of 55 isolates from 30 patients were identical by PFGE analysis after SmaI and SacII digestion and immunoblot analysis. Two isolates were different from each other and from the epidemic group by both PFGE and immunoblot analysis. All 53 epidemic isolates were nontoxigenic, while the two remaining isolates were toxigenic. These results suggest that nosocomial spread of nontoxigenic C. difficile infection in the neonatal intensive care unit and suggest that both PFGE and immunoblot are powerful typing tools for the epidemiological study of C. difficile.