The persistence and ability of transformed Lactobacillus strains to function and produce β-glucanase in the gastrointestinal tract of chickens was investigated. Chickens were fed diets supplemented with 10⁹ cells/kg of transformed Lactobacillus strains which harboured plasmid pSA3 carrying a gene encoding β-glucanase. The transformed β-glucanase-producing Lactobacillus strains were L. brevis C10CpSA3b6, L.crispatus I12pSA3b6, L. brevis I23pSA3b6, L. fermentum I25pSA3b6, L. brevis I211pSA3b6 and L. brevis I218pSA3b6. At days 14 to 18, digesta samples from the gizzard, duodenum, jejunum, ileum and caecum were collected daily and the presence of the respective β-glucanase-producing Lactobacillus strains was determined by a PCR-based method. Results of the study showed that the β-glucanase-producing Lactobacillus strains could persist in the gastrointestinal tract of the chickens for about two days. The strains were found to be alive and active in vivo, in which β-glucanase activity was significantly (P<0.05) higher in chickens fed diets supplemented with β-glucanase-producing Lactobacillus strains. Higher enzyme activities were located in the jejunum and ileum, especially in birds fed β-glucanase-producing L. brevis C10CpSA3b6 and L. brevis I211pSA3b6. The results indicated that the Lactobacillus strains could be a potential vehicle to deliver enzymes into the gastrointestinal tract of chickens.