The effect of four sugars (glucose, galactose, lactose and fructose) on exopolysaccharide (EPS) production by Bifidobacterium longum subsp. longum CRC 002 was evaluated. More EPS was produced when CRC 002 was grown on lactose in the absence of pH control, with a production of 1080±120 mg EPS l−1 (P<0.01) after 24 h of incubation. For fructose, galactose and glucose, EPS production was similar, at 512±63, 564±165 and 616±93 mg EPS l−1, respectively. The proposed repeating unit composition of the EPS is 2 galactose to 3 glucose. The effect of sugar and fermentation time on expression of genes involved in sugar nucleotide production (galK, galE1, galE2, galT1, galT2, galU, rmlA, rmlB1 and rmlCD) and the priming glycosyltransferase (wblE) was quantified using real-time reverse transcription PCR. A significantly higher transcription level of wblE (9.29-fold) and the genes involved in the Leloir pathway (galK, 4.10-fold; galT1, 2.78-fold; and galE2, 4.95-fold) during exponential growth was associated with enhanced EPS production on lactose compared to glucose. However, galU expression, linking glucose metabolism with the Leloir pathway, was not correlated with EPS production on different sugars. Genes coding for dTDP-rhamnose biosynthesis were also differentially expressed depending on sugar source and growth phase, although rhamnose was not present in the composition of the EPS. This precursor may be used in cell wall polysaccharide biosynthesis. These results contribute to understanding the changes in gene expression when different sugar substrates are catabolized by B. longum subsp. longum CRC 002.