It is difficult in some cases to identify an infection caused by Borrelia burgdorferi and to monitor the effect of therapy. Seropositivity will persist even after successful treatment and therefore may suggest ongoing infection, For direct detection of B, burgdorferi DNA in human urine samples, the polymerase chain reaction (PCR) was evaluated, A published primer system was selected, which amplifies a 259 bp fragment from the gene encoding the 23S rRNA. The lower detection limit of the primer system was 10 fg of extracted B, burgdorferi DNA, Several methods for the pretreatment of urine samples were tested, Of these, the Geneclean(R) kit (Bio 101, USA) showed the best results; A total of 114 urine samples from 74 patients belonging to three clinical groups was investigated: (i) 51 samples from 26 patients with active Lyme disease, (ii) 36 samples from 27 patients with previous infection but no symptoms at the time the urine was collected, and (iii) 27 samples from 21 seronegative control patients without Lyme disease, B, burgdorferi DNA was detected in 25 urine samples of 17 patients with active disease, whereas 26 samples from this group of patients were negative, Only one asymptomatic case with previous infection showed a positive result, and the urine samples of the patients without Lyme disease were uniformly negative, Two of four patients from whom samples before and directly after onset of therapy were available converted from negative to positive PCR results after initiation of therapy, accompanied by the symptoms of a Jarisch-Herxheimer reaction, It can be concluded from these results that a positive PCR from urine is,vith high probability an indicator of active Lyme disease, On the other hand, as only 17 of the 26 patients with active infection were positive, a negative PCR result does not exclude active infection.