Piliated Neisseria gonorrhoeae forming small, transparent colonies (P+O-) on clear typing agar have been grown in prolonged continuous culture to ascertain how different growth environments might affect gonococcal physiology and the expression of virulence determinants. Virulence of the penicillin-sensitive P9-2 and the penicillin-resistant KW1 strains was assessed by their ability to survive in polypropylene chambers implanted into the flanks of guinea pigs. Initial continuous culture experiments in the defined medium of Manchee et al. (FEMS Microbiology Letters 7, 115-118, 1980) indicated that growth was actually cystine-limited, rather than the anticipated glucose-limited. Surprisingly, cysteine was not completely metabolized and ammonium salts remained in excess. The molar growth yield on glucose (YGlc) was 65 g dry wt mol-1 and 45% of the glucose carbon metabolized was converted to biomass. Gonococci, whilst retaining the P+O- phenotype for over 100 generations of growth, did not survive in the subcutaneous chambers when inoculated at a variety of doses. When the cystine and glucose concentrations were increased and decreased respectively, growth became glucose-limited, the YGlc increased to 108 g mol-1 for strain KW1 and 75% of the metabolized glucose carbon was converted to biomass. After 17 generations of growth, however, only 2% of the gonococci retained the P+O- phenotype and P-O- bacteria predominated. Nevertheless, these bacteria were virulent in the chamber model, as was strain P9-2, which also retained only 2% of the P+O- phenotype during glucose-limited continuous culture. By contrast, the P+O- phenotype was retained during prolonged cystine- or oxygen-limited growth but only the latter was virulent. SDS-PAGE of membrane extracts confirmed that opaque colonies (O+) selected from the glucose-limited cultures contained a heat-modifiable protein (protein II) whereas transparent colony types lacked such proteins. The initial phenotype of virulent gonococci recovered from the subcutaneous chambers was P+O- but opaque variants dominated after several days. A 40 kDa outer-membrane protein was apparently induced during oxygen-limited continuous culture whereas a 44 kDa protein was absent during cystine-limited growth.