In this study, the regularity of autoinducer-2 (AI-2) secretion during growth and the effect of the addition of various carbohydrates on AI-2 secretion in Bifidobacterium longum subsp. longum BBMN68 were investigated. The results indicated that the AI-2 concentration reached its highest level (2536.60 nmol l-1 ) at the early stationary growth phase, and then decreased to 1263.72 nmol l-1 at the late stationary growth phase in Bifidobacterium cultures. When the density of the cultures which mannose, fructose, sucrose, and lactose had been added to reached an OD600 nm of 1.0, the AI-2 concentrations in the cultures were 1953.84, 1637.34, 1200.99 and 1077.60 nmol l-1 , respectively. These concentrations were all significantly higher than that of the control culture (1031.33 nmol l-1 ). Similarly, the addition of fructooligosaccharide significantly increased the AI-2 concentrations to 2094.29 nmol l-1 . This study provides the advanced evidence that certain carbohydrates promote the secretion of AI-2, and that this occurs at the single cell level and is therefore unaffected by cell density. This article is protected by copyright. All rights reserved.