In previous work, a factor which enhances the ability of cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) to sialylate gonococcal lipopolysaccharide (LPS) was liberated at 4 degrees C in diffusates from high M(r) fractions of blood cell sonicates. The diffusates also contained CMP-NANA and converted serum susceptible gonococci to resistance. The enhancer has now been separated from CMP-NANA and material absorbing at 260 nm by HPLC on mu Bondapak-10 NH2. Resistance inducing activity was found only in fractions containing CMP-NANA and recovery was poor (about 25%). However, addition of enhancer fractions to CMP-NANA substantially increased its resistance inducing activity. Blood cell sonicates dialysed at 18-20 degrees C released enhancer in diffusates. These were ultrafiltered (nominal cut off 3000 Da) and fractionated on Biogel P2 which removed saccharides and most material absorbing at 260 nm. Over 90% of a fraction which was enhancer-active in nanogram quantities was identified by nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectometry (GC/MS) as lactic acid. A fraction with similar properties was obtained from a different batch of diffusate by fractionation on Dowex 1. Authentic lithium L-lactate in nanogram quantities enhanced LPS sialyation by CMP-NANA and increased its serum resistance inducing activity. These results have important implications for gonococcal pathogenicity.