Longterm, strictly anaerobic proliferation of three laboratory strains of B. burgdorferi was achieved, when medium OMIZ-WP was modified by increasing the concentrations of sodium bicarbonate and potassium chloride and by adding 5-10 per cent fetal bovine serum (FBS). Growth was strictly dependent on the presence of at least one of the two chemically complex components of OMIZ-WP, i.e. a methanol-soluble fraction of yeast extract (YEM) or de-anionised Neopeptone (DANP). By fractionation of DANP we disclosed two components essential for in vitro proliferation of B. burgdorferi. One of these could be replaced by spermidine. The other was isolated and identified as adenine. No other polyamines or purines could replace spermidine and adenine, respectively. This information allowed formulation of a chemically defined medium supporting proliferation of B. burgdorferi in the presence of 10 per cent FBS. Further, as yet unidentified components of DANP and/or YEM are, however, required to support growth to higher cell densities.