Clostridium difficile strain NAP1/027 (North American pulsed-field gel electrophoresis [PFGE] type 1 and PCR ribotype 027 [R027]) has been associated with recent outbreaks in North America and Europe. It has been associated with more severe disease symptoms, higher mortality rates, and greater risk of relapse. This strain is thought to produce more toxins and sporulate to higher levels. However, recent studies suggest that this may not always be the case. The objective of our study was to assess, in a nonoutbreak situation, whether specific strains, such as NAP1/027, were associated with more severe disease symptoms, higher toxin production, and/or greater sporulation in vitro. We isolated and characterized C. difficile strains from 21 patients with mild to moderate, severe, or complicated symptoms of C. difficile infection (CDI). The isolates were characterized by different molecular typing methods, including PCR ribotyping, tandem repeat sequence typing (TRST), and sequencing of the tcdC gene. Fourteen isolates were of PCR ribotype 027 with deletions in tcdC, but no association with severity or clinical outcome was found. We show by immunodot blot detection of toxins with monoclonal antibodies that all R027 isolates produced more TcdA and TcdB than other strains. On the other hand, they consistently produced fewer spores than non-R027 isolates. Taken together, our data suggest that NAP1/027 isolates are not always associated with more severe disease, even though they may produce larger amounts of toxins. Our study also suggests that current assertions regarding the NAP1/027 may not apply to all isolates and that other factors may come into play.