Conventional and real-time PCR were used to detect transgenic DNA in digesta, faeces and blood collected from six ruminally and duodenally cannulated sheep fed forage-based (F) or concentrate-based (C) diets containing 15 % Roundup Ready® (RR) rapeseed meal (n 3). The sheep were adapted for 14 d to F or C diets containing non-GM rapeseed, then fed the RR diets for 11 d. On day 12, they were switched back to non-GM diets for a further 11 d. Ruminal and duodenal fluids (RF, DF) and faecal samples were collected at 3 or 4 h intervals over the 4 d immediately following the last feeding of GM diets. DNA was isolated from whole RF and DF, from the cell-free supernatant fraction, and from culture fermentation liquid. Blood was collected on days 1, 5 and 9 of feeding the RR rapeseed meal. The 1363 bp 5-enolpyruvylshikimate-3-phosphate synthase transgene (epsps) was quantifiable in whole RF and DF for up to 13 h, and a 108 bp epsps fragment for up to 29 h. Transgenic DNA was not detectable in faeces or blood, or in microbial DNA. Diet type (F v. C) did not affect (P>0·05) the quantity of transgenic DNA in digesta. More (P < 0·05) transgenic DNA was detected in RF than in DF, but there was an interaction (P < 0·05) between sample type and collection time. In supernatant fractions from RF and DF, three different fragments of transgenic DNA ranging in size from 62 to 420 bp were not amplifiable.