Complex media are commonly used in studies examining exopolysaccharide production by Lactobacillus delbrueckii ssp. bulgaricus. However, quantification of exopolysaccharide in complex medium can be complicated by interference due to carbohydrate polymers contained in media components. This study was undertaken to identify components of MRS, a common medium for cultivation of L. delbrueckii ssp. bulgaricus, that interfere with exopolysaccharide quantification, to develop a medium for production of exopolysaccharide that provides for growth of L. delbrueckii ssp. bulgaricus strain RR similar to MRS, and to demonstrate exopolysaccharide production by L. delbrueckii ssp. bulgaricus RR grown in the newly developed medium. Phenol-sulfuric acid determinations were conducted on uninoculated MRS broth with and without yeast extract, beef extract and proteose peptone #3. These three ingredients accounted for 94% of the total background exopolysaccharide-equivalent in MRS broth. Based on these results, a semi-defined medium (SDM) providing minimal interference was developed using yeast nitrogen base and Bacto casitone. Growth of L. delbrueckii ssp. bulgaricus RR at 42 degrees C in semi-defined medium and MRS was evaluated, and generation times did not differ significantly (0.94 h in MRS and 0.85 h in SDM). Exopolysaccharide production by L. delbrueckii ssp. bulgaricus RR during growth in semi-defined medium was evaluated at 30 and 40 degrees C. The rate of exopolysaccharide production was lower at 30 degrees C (8.04 (mg/l-h) than at 40 degrees C (11.95 (mg/l-h), but the maximal concentration of exopolysaccharide produced was similar at both temperatures.