BACKGROUND: A study was conducted among newly registered HIV-positive tuberculosis (TB) patients systematically offered anti-retroviral treatment (ART) in a district hospital in rural Malawi in order to a) determine the acceptance of ART b) conduct a geographic mapping of those placed on ART and c) examine the association between "cost of transport" and ART acceptance. METHODOLOGY/PRINCIPAL FINDINGS: A retrospective cross-sectional analysis was performed on routine program data for the period of February 2003 to July 2004. Standardized registers and patient cards were used to gather data. The place of residence was used to determine road distances to the Thyolo district hospital. Cost of transport from different parts of the district was based on the known cost for public transport to the road-stop closest to the patient's residence. Of 1,290 newly registered TB patients, 1,003(78%) underwent HIV-testing of whom 770 (77%) were HIV-positive. 742 of these individuals (pulmonary TB = 607; extra-pulmonary TB = 135) were considered eligible for ART of whom only 101(13.6%) accepted ART. Cost of transport to the hospital ART site was significantly associated with ART acceptance and there was a linear trend in association between cost and ART acceptance (chi(2) for trend = 25.4, P<0.001). Individuals who had to pay 50 Malawi Kwacha (1 United States Dollar = 100 Malawi Kwacha, MW) or less for a one-way trip to the Thyolo hospital were four times more likely to accept ART than those who had to pay over 100 MW (Adjusted Odds ratio = 4.0, 95% confidence interval: 2.0-8.1, P<0.001). CONCLUSIONS/SIGNIFICANCE: ART acceptance among TB patients in a rural district in Malawi is low and associated with cost of transport to the centralized hospital based ART site. Decentralizing the ART offer from the hospital to health centers that are closer to home communities would be an essential step towards reducing the overall cost and burden of travel.