Assays employing iron-limited solid and liquid, defined and complex media were devised to test the iron requirements of Neisseria meningitidis. A variety of tests yielded no evidence for the secretion of a soluble iron-binding substance (siderophore) by the meningococci. The meningococci were unable to use iron bound to some common hydroxamate- and catechol-type siderophores or even compete with them for iron in the growth medium. A total of 20 strains of meningococci, differing widely in their virulence for mice, were similar in ability to acquire iron from a variety of iron-containing substances; the iron in such compounds as hog gastric mucin, citrate, hemoglobin, and myoglobin was easily acquired, whereas the iron in compounds such as ferrioxamine B, ferrichrome,ferritin, Imferon, cytochrome c, FePO4, and [Fe(OH)3]n was not readily available. No correlation was noted between the ability of particular strains to obtain iron from compounds and virulence in mice. Iron complexed or chelated with a number of metabolic organic acids, polyphosphates, and several synthetic polycarboxylic acids was readily available to all strains, even though some of the compounds used had high effective binding constants for iron and all were in 3- or 10-fold molar excess over the iron present. The addition of some of these iron-complexing substances (e.g., citrate and pyrophosphate) in iron-free form made many biologically important iron compounds that are normally inaccessible to the meningococci readily available.