Kefir is a fermented milk drink produced by kefir grain. Kefir grains are used as the natural starter in kefir manufacturing and consist of a variety of microorganisms such as lactic acid bacteria, yeasts and acetic acid bacteria. Peptides and exopolysaccharide are formed during fermentation that has been shown to have bioactive properties such as anti-carcinogenic, anti-mutagenic, anti-viral and anti-fungal properties. For optimization of EPS production, the effect of different conditions (strains, temperature, different sugar at different concentration and nitrogen sources) were investigated in pure cultures of the isolated microorganism by microplate titration method. Also the best conditions in this experiment were used to produce functional kefir. Different properties of produced kefir such as pH, acidity, viscosity, fat, dry material, microbial population and EPS was compared with the kefirs produced by pure culture of isolated microorganism. Also the effect of different conditions in kefir grain production was studied. Data of enumeration of microorganism showed that the population of lactobacillus were 10 7 -10 8 CFU/ml, lactoccocus 10 8 -10 9 CFU/ml, yeast 10 6 -10 7 CFU/ml and acetobacter were 10 5 CFU/ml in kefir. Identification of the isolated microorganism with biochemical tests showed that the isolated microorganism were Lactobacillus delbreuckii su bulgaricus , Lactobacillus delbreuckii su lactis, Entrococcus facium, lactococcus lactis, Acetic acid bacteria and Yeast. Lactobacillus delbreuckii su bulgaricus was the best strain for EPS production and a chemically defined medium with 4% lactose, 2%-3% pepton casein and incubation at 37 ?C with 5% CO 2 was the best culture condition for EPS production. Production of EPS in kefir has been significantly increased by addition of 1% lactose, 1% Lactobacillus delbreuckii su bulgaricus and 37 o C incubation (P 0.05). In this functional kefir acidity, pH, viscosity and microbial population of lactobacillus and yeast have been changed significantly, however fat, dry material and population of coccus did not have significant changes. Addition of pure cultures of the isolated bacteria instead of kefir grains produced high EPS and viscosity (3067 cp). The conditions, using 1.5 ml of 10% skim milk without adding fat, addition of 2% glucose,1% yeast extract, incubation at 37 ?C and presence of 10% CO 2 were also the optimum conditions for kefir grain production which suggested that EPS producing bacteria are responsible for kefir grain production.