Optimization of the cultivation conditions and medium components for folate production by the highest folate producing Streptococcus thermophilus strain RD102 was carried out using a 23 central composite design and surface modelling method of response surface methodology. As folate production was observed to be growth-associated, hence the desired responses selected for the optimization were both folate production and growth. The selected factors based on preliminary investigations were incubation period, concentrations of p-amino benzoic acid (PABA) the growth precursor, and lactose as carbon source. The optimum concentrations of PABA and lactose were found to be 300 micropm and 3% respectively at 72 h incubation. The optimized conditions resulted in an increase in folate production by 26% compared with control conditions (0% PABA and lactose at 37 degrees C). Using the method of experimental factorial design and response surface analysis, it was possible to determine optimal operating conditions to obtain a higher folate production by Strep. thermophilus. Therefore, this study constitutes a step in developing strategies to modulate the folate level to a higher level.