BACKGROUND:  The impact of glyphosate on ruminal fermentation, selective pressure on ruminal bacteria and horizontal transfer of the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (epsps) to ruminal bacteria was studied using batch culture with glyphosate-tolerant (Roundup Ready®) canola meal as substrate. RESULTS:  A glyphosate concentration × time interaction (P < 0.05) occurred when glyphosate (0-100 mmol L-1) was included in in vitro ruminal incubations with a diet containing 150 g kg-1 Roundup Ready® canola meal (Experiment 1).  Glyphosate at 50 and 100 mmol L-1 inhibited fermentation.  In Experiment 2, epsps fragments were detected in plant debris for up to 16 h of incubation using primer sets that amplified three fragments (62, 108 and 300 bp) of DNA spanning the transgenic construct.  Persistence was affected by fragment size but not by glyphosate concentration (0, 10 or 60 mmol L-1).  Extensive polymerase chain reaction assays provided no evidence of acquisition of epsps by feed- or fluid-associated bacteria during fermentation.  A glyphosate concentration × time interaction (P < 0.05) was observed for all fermentation parameters measured, and glyphosate caused a general inhibition of fermentation. CONCLUSION:  The presence of glyphosate did not increase selective pressure for gene transfer of DNA encoding glyphosate resistance from Roundup Ready® canola meal to ruminal bacteria.