Aims: To evaluate the behaviour of Shiga toxin-producing Escherichia coli (STEC) O26 strains inoculated in manure-amended soils under in vitro conditions. Methods and Results: Four green fluorescent protein (GFP)-labelled STEC O26 strains were inoculated in duplicate (at 10(6) CFU g(-1)) in three different manure-amended soil types, including two loam soils (A and B) and one clay loam soil (C), and two incubation temperatures (4 and 20 degrees C) were tested. STEC counts and soil physical parameters were periodically monitored. STEC O26 cells were able to persist during extended periods in soil even in the presence of low moisture levels, i.e. less than 0.08 g H2O g(-1) dry soil. At 4 and 20 degrees C, STEC could be detected in soil A for 288 and 196 days, respectively, and in soils B and C for at least 365 days postinoculation at both temperatures. The ambient temperature (i.e. 20 degrees C) was significantly associated with the highest STEC count decline in all soils tested. Conclusions: The temperature and soil properties appear to be contributory factors affecting the long-term survival of STEC O26 in manure-amended soils. Significance and Impact of the Study: This study provides useful information regarding the ecology of STEC O26 in manure-amended soils and may have implications for land and waste management.