Haemophdus influenme is a V factor-dependent species. A plasmid conferring V factor independence in Haemophilus parainfluenme and Haemophilus ducreyi was transferred to plasmid-free H. influenzae Rd by DNA transformation. The growth characteristics of the transformants in a complex and a chemically defined medium were compared, and the ability of several exogenous pyridine nucleotides and precursors to support growth was examined. Although the transformants appeared to be V factor independent in a complex medium, in a chemically defined medium they exhibited both V factor-dependent and nicotinamide-dependent growth. Because of the inability of the plasmid-free H. influenzae Rd to utilize nicotinamide for growth, it was concluded that the genes conferring this function were plasmid linked. Our results indicate that the V factor requirement, as it is presently defined, is not suitable to serve as a definitive taxonomic criterion for species determination in the family Pasteurekeae. Haemophilus influenzae, Haemophilus parainfluenzae, and several other members of the genus Haemophilus re- quire V factor (P-nicotinamide adenine dinucleotide "AD)) for growth (11, 13). Unlike most bacteria, these species are not able to synthesize NAD de novo from low-molecular- weight compounds but use a limited number of exogenous pyridine nucleotides or precursors as a source of NAD. Previous studies have shown that in addition to NAD, nicotinamide riboside (NR), P-nicotinamide mononucleotide (NMN), and NADP can serve as a V factor, while nicoti- namide (NAm), nicotinic acid (NA), and quinolinic acid (QA) cannot (3, 4). The V factor requirement and the requirement for X factor have been used for many years as a basis for the classification and the laboratory identification of species from the genus Haemophilus. Four unusual naturally occurring V factor-independent H. parainfluenzae isolates were identified in our laboratory a few years ago (5, 6). The genes coding for V factor indepen- dence were located on a small 5.25-kb plasmid (24). Plasmids of a similar size conferring the ability to grow in the absence of V factor were recovered recently from Haemophilus ducreyi (25). This species is presently classified as a V factor-independent Haemophilus species, but DNA homol- ogy studies and physiological differences between H. du- creyi and other Haemophilus species have recently been used to question its inclusion as a Haemophilus species. In this report we present data on the growth characteris- tics of H. influenzae Rd transformants in the presence of a plasmid conferring V factor independence. Attempts were made to determine whether the plasmid confers the ability to utilize an exogenous pyridine nucleotide precursor (such as NAm) for growth. It is known that NAm can be used as a source of NAD by other V factor-independent members of the genus Haemophilus (10). Because of the extrachromo- soma1 location of the genes coding for V factor indepen- dence, we conclude that this characteristic is not suitable to serve as a major species-determining taxonomic criterion.