C9 is required for rapid complement-mediated killing of E. coli by serum. Previous studies revealed that a diminished C9 concentration restricts the bactericidal capacity of serum from neonatal humans and neonatal rats. Also, significant morbidity and mortality from E. coli sepsis persist in neonates despite the use of bactericidal antibiotics, such as cefotaxime (CEF). Therefore, in vitro and in vivo experiments were designed to determine whether supplemental C9 potentiates the bactericidal and protective effects of CEF in neonatal rats. Undegraded and hemolytically active human C9 was purified from Cohn fraction III paste. First, the survival of E. coli (5 × 104 cfu/ml) was quantitated in vitro during 60 min of incubation with 20% serum pooled from neonatal rats (NeoRS). The bacteria proliferated in NeoRS(bacterial survival [x±SEM] = 199±15% of the original inoculum). Similarly, the bacteria proliferated in NeoRS supplemented with 2.5 ng/ml CEF(bacterial survival = 170±26%) or with 20 μg/ml C9 (147±11%). In contrast, only 54±27% of the bacteria survived in serum supplemented with both CEF and C9 (P<.04 vs unsupplemented NeoRS, vs NeoRS+CEF, and vs NeoRS+C9). Next, studies were conducted in vivo. Two-day-old rats received transthoracic injections of E. coli (1.7× 106/g body wt [bw]). One h later, quantitative cultures revealed that the blood of 15/16 animals contained E. coli(1611±537 cfu/ml). By 4 h, the blood of all animals injected containedE. coli. Next, the survival of neonatal rats was quantitated one wk after transthoracic injections of E. coli. The bacteria were injected into each neonate immediately after the subcutaneous (sc) administration of CEF (15 ng/g bw) or an equal volume of placebo (PBS). Each animal also received intraperitoneal (ip) C9 (75 μg/g bw) or ip PBS. 43 animals received only sc and ip PBS prior to the injection of bacteria. 22 of these 43 controls survived. 11/44 CEF recipients and 12/44 C9 recipients survived (P>.11 vs recipients of only PBS). In contrast, 36/45 recipients of both CEF and C9 survived (P<.05 vs only PBS, vs CEF alone, and vs C9 alone). In conclusion, supplemental C9 potentiated the bactericidal activity of CEF against E. coli in NeoRS and potentiated the protective effect of CEF in septic neonatal rats. These observations may contribute to the development of new strategies to reduce the morbidity and mortality associated with E. coli sepsis in human neonates. Funded by the Alliant Community Trust Fund.