In humans, gonococcal infection occurs in environments limited with respect to free iron. Neisseria gonorrhoeae produces increased quantities of iron-regulated membrane proteins when grown under in vitro conditions which restrict the availability of free iron. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) techniques, we studied the reactivity of specific antibodies to the 37-kilodalton (kDa) major iron-regulated protein (MIRP) of gonococci grown under iron-limiting conditions. Antibodies reactive with the 37-kDa MIRP were distinguished from those reactive with protein I by using purified 37-kDa MIRP or gonococcal protein preparations. Acute-phase sera from patients with disseminated gonococcal infection (DGI) reacted strongly to both the 37-kDa MIRP and protein I. Acute sera from nine patients with uncomplicated gonorrhea did not exhibit strong reactivity with the 37-kDa MIRP and were indistinguishable from five control sera. When compared with acute-phase sera, convalescent-phase sera from patients with DGI failed to demonstrate increased reactivity, whereas convalescent-phase sera from one of nine patients with uncomplicated gonorrhea developed reactivity to the 37-kDa MIRP. These data indicate that (i) the 37-kDa MIRP is expressed and antigenic in vivo and (ii) humans with DGI consistently develop a systemic antibody response to the 37-kDa MIRP.