We examined the hypothesis that the sac-4 gene of Neisseria gonorrhoeae varies with gonococcal subtype and that this could account for an earlier report that sac-4 increased the likelihood of co-infection with Chlamydia trachomatis. A polymerase chain reaction (PCR) assay was used to determine the prevalence of sac-4 in 435 gonococcal isolates. The prevalence of sac-4 was analysed in relation to chlamydial co-infection, serovar, auxotype, gender and sexual orientation. Although the prevalence of sac-4 was higher in isolates from patients with chlamydial co-infection (55%) than in those without chlamydial co-infection (42%) the difference was not significant (P<0.05). Statistically significant differences in association with sac-4 were, however, shown between various serovars and auxotypes. Dual classification based on auxotype/serovar (A/S) classes showed highly significant differences in sac-4 prevalence between groups: 95% in NR/1B18 and 8% in P/1B2 (P<0.001). Sac-4 was also significantly less common (P<0.05) in isolates from homosexual men (35%) than from heterosexual men (49%) or women (49.5%). Sac-4 appears to have an epidemiological association with gonococcal auxotype and serovar rather than a direct association with chlamydial co-infection.