In Thyolo district, Malawi, an operational research study is being conducted on the efficacy and feasibility of co-trimoxazole prophylaxis in preventing deaths in HIV-positive patients with tuberculosis (TB). A series of cross-sectional studies were carried out in 1999 and 2001 to determine (i) whether faecal Escherichia coli resistance to co-trimoxazole in TB patients changed with time, and (ii) whether the resistance pattern was different in HIV-positive TB patients who were taking co-trimoxazole prophylaxis. Co-trimoxazole resistance among E. coli isolates in TB patients at the time of registration was 60% in 1999 and 77% in 2001 (P < 0.01). Resistance was 89% among HIV-infected TB patients (receiving cotrimoxazole), while in HIV-negative patients (receiving anti-TB therapy alone) it was 62% (P < 0.001). The study shows a significant increase of E. coli resistance to co-trimoxazole in TB patients which is particularly prominent in HIV-infected patients on co-trimoxazole prophylaxis. Since a high degree of plasmid-mediated transfer of resistance exists between E. coli and the Salmonella species, these findings could herald limitations on the short- and long-term benefits to be expected from the use of co-trimoxazole prophylaxis in preventing non-typhoid Salmonella bacteraemia and enteritis in HIV-infected TB patients in Malawi.