In this study, three conjugated linoleic acid (CLA)-producing strains of Lactobacillus were cultured up to 36 in reconstituted skim milk powder (10%) supplemented with 2000 μg/ml of free linoleic acid (LA) and various concentrations of Tween 80. During the incubation, total CLA levels in the culture supernatants were determined by UV-spectrophotometry. The CLA levels significantly (P<0.05) increased with the addition of 5 and 20 mg/ml Tween 80. However, increasing Tween 80 concentration from 20 to 40 mg/ml did not appear to enhance CLA levels. Similar increase patterns were observed in the growth rate and CLA production of Lactobacillus strains during the incubation.