The present investigation was carried out in order to obtain better information about the growth requirements for small-sized oral spirochetes containing two endoflagella from each cell-end. Eight strains of such spirochetes were isolated from subgingival plaque in patients suffering from advanced marginal periodontitis. The strains were maintained under anaerobic conditions in a fluid basal BHI medium with 15% inactivated rabbit serum, 0.07% Noble Agar and 5 micrograms/ml cocarboxylase. Firstly, the effect of trace amounts of oxygen in the atmosphere and pH in the medium on growth of the spirochete strains were examined. Secondly, the effect of different sera incorporated in the medium was examined, and thirdly, the effect of important growth factors in serum was studied by adding different serum components to the fluid basal medium instead of rabbit serum. Growth was always determined after 4 days' incubation at 35 degrees C, either by counting numbers of spirochete cells in a Petroff-Hauser counting chamber or by measuring the turbidity of the culture spectrophotometrically at 600 nm. There was no difference in growth by using an atmosphere containing 1% oxygen or an anaerobic atmosphere. It was found that serum (rabbit or human) was an essential growth component, and no single growth factor could replace rabbit serum. Only a long chain fatty acid mixture and an amino acid solution could, to a minor extent, stimulate growth compared to the basal medium without rabbit serum. Sodium bicarbonate inhibited growth of all strains. Finally, none of the strains fermented a series of low molecular weight carbohydrates, but all strains produced H2S and indole.