The virulence mechanism of Neisseria gonorrhoeae in pelvic inflammatory disease (PID) is not well understood, and an objective diagnostic method to identify patients with PID is lacking. We investigated the hypothesis that development of PID was associated with a C1q-dependent virulence property of gonococcal strains. Recent development of a C1q-dependent experimental model of gonococcal infection (S. Nowicki, M. Martens, and B. Nowicki, Infect. Immun. 63:4790-4794, 1995) created an opportunity to evaluate this hypothesis in vivo. Therefore, the virulence of 32 clinical isolates (18 PID isolates and 14 local infection [LI] isolates) was evaluated in experimental rat pups. A serum bactericidal assay was used to characterize a gonococcal serum-resistant (ser(r)) phenotype. PCR primers designed to amplify a suitable-size gonococcal sac-4 DNA fragment (unique for serum-resistant donor JC1) were used to evaluate the association of serum-resistant genotype sac-4 with two phenotypes: C1q-dependent virulence expressed in vivo and resistance to bactericidal activity of human serum expressed in vitro. Strains were also characterized by auxotyping and serotyping. Of 32 gonococcal strains, 15 (46.7%) caused C1q-dependent bacteremia in rat pups and were sac-4 positive and ser(r). However, of the 15 isolates, 13 (87%) represented strains associated with human PID and 2 (13%) were associated with LI. None of the strains that were completely serum-sensitive (ser(s)) and sac-4 negative produced C1q-dependent bacteremia in rat pups, suggesting that both ser(r) and sac-4 were required for infection. The serum-resistant recombinant recipient of sac-4 produced C1q-dependent bacteremia in the rat model similarly to the serum-resistant donor of sac-4; the serum-sensitive parent strain did not produce bacteremia. These data suggest that sac-4-mediated serum resistance conferred C1q-dependent virulence and is a unique characteristic associated with PID. These newly identified features may contribute to the understanding of the pathogenic mechanism of PID-associated strains and open perspectives for establishing novel diagnostic methods.