The purpose of this study was to investigate the usefulness of topical provocation in the diagnosis of cotrimoxazole-induced fixed-drug eruption (FDE). 27 patients with established cotrimoxazole-induced FDE by oral provocation and 20 healthy controls were tested with drugs at increasing concentrations in white petrolatum and dimethyl sulfoxide (DMSO) both on previously involved and uninvolved skin sites. Tape-stripping occlusive patch testing in petrolatum remained negative in 19 tested patients. Open testing with drug preparations in DMSO revealed positive results in 25 of 27 tested patients. I patient showed an additional positive reaction on previously uninvolved skin. Lesions on male genitalia and on face reacted to testing once with 10% or 20% of the suspected drug, whereas repeated testing with concentrations up to 50% was necessary in lesions on trunk & extremities. Open testing with drug preparations in DMSO at concentrations of 10%, 20% and 50% and pure DMSO remained negative in 20 healthy controls. The present study shows that repeated open testing with graded concentrations of the drugs up to 50% in DMSO is a reliable test method in sulfamethoxazole/trimethoprim-induced FDE. Patients and physicians should be aware of the transient irritant reaction to DMSO that is not infrequent, so as to avoid false-positive interpretations.