Haemophilus influenzae has an absolute requirement for factor V because it lacks all the biosynthetic enzymes necessary for the de novo synthesis of NAD. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mono-nucleotide (NMN) or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake for factor V in vivo. Recently, a hypothetical open reading frame (ORF), termed nadN, was identified to encode a gene product essential for H. influenzae growth on NAD. Here, we report its role in the virulent H. influenzae serotype b strain Eagan. Our results indicate that NadN of type b Eagan strains is involved in NAD uptake and in processing NAD to NR, which appears to be the substrate for an as yet unidentified cytoplasmic membrane NR transport system. Furthermore, we present data showing that H. influenzae type b nadN mutants are able to survive as well as Eagan, in vivo in the five-day-old infant rat model of human invasive disease. NAD pyrophosphatase and NMN 5'-nucleotidase activities were present in rat and human serum, implying that under infection conditions H. influenzae may obtain NR directly from its host.