Background: We recently observed that a short course of trimethoprim 300 mg b.i.d. in healthy volunteers can cause a substantial increase in fasting plasma homocysteine levels, up to concentrations reportedly associated with atherothrombotic complications. The purpose of this study was to determine whether primary Pneumocystis carinii prophylaxis (PCP) with trimethoprim-sulphamethoxazole (TMP-SMX) adversely affects serum homocysteine levels in HIV-positive patients. Methods: We studied 34 subjects [29 male, 5 female, mean age 36.8±7.9 (S.D.) years] with no prior AIDS-defining disease who required primary PCP prophylaxis (CD4+ T-cell count <200/mm3). The common dose of TMP-SMX was 80/400 mg (80 mg trimethoprim and 400 mg sulphamethoxazole) once daily. Serum total homocysteine levels were determined in four samples: two collected prior to the start of TMP-SMX and two collected on average 2.6±2.2 and 5.3±3.5 months into the first year of prophylactic therapy. Results: Mean serum homocysteine was 13.9±3.7 μmol/l pre-treatment and 14.4±5.0 μmol/l during treatment with TMP-SMX, a non-significant increase of 0.5 μmol/l (95% CI: -0.5 to +1.4, P=0.34). Folate levels were equally unaffected by TMP-SMX (13.1±6.5 nmol/l versus 13.3±5.3 nmol/l, before and during therapy, respectively). Baseline folate levels did not predict the response of homocysteine to TMP-SMX, and neither did age, gender, or serum creatinine. Conclusion: Long-term therapy with 80/400 mg TMP-SMX does not adversely affect homocysteine levels.