The severity of the clinical course in necrotizing enterocolitis (NEC) associated with Clostridium perfringens (Cp) may support the hypothesis of a specific disease. We conducted a case control study of infants diagnosed with NEC, who underwent surgical treatment over a 7-year period. Patient histories examined characteristics of the infants, bacterial infection as well as NEC's severity, antibiotic treatment, and clinical course. Infants infected with NEC associated with Cp were compared with NEC patients without Cp. The alpha toxin from Cp type A was detected in most of the isolated strains. Cp was identified as a causative agent of NEC in nine cases. As compared with the control group (n = 32), the onset of disease was earlier in life, the clinical course more severe, and patients had a larger extent of gangrene. Portal venous gas was evident in 77% of all Cp cases, as compared to 25% in the control group. The mortality rate was 44% in the Cp group, and only 18.7% in the control group. Type A Clostridium perfringens was identified in six cases. In each isolate alpha toxin production was proven, but without any correlation to the severity of the clinical course, the extent of intestinal gangrene or mortality. In premature infants NEC in conjunction with Cp seems to be more severe than other NEC cases; it also entails higher mortality and morbidity. Alpha toxin concentrations do not correlate with the severity of the disease. Portal venous gas is highly suggestive for the diagnosis of Cp infection.