Lyme borreliosis is a disease caused by spirochaetes belonging to the genospecies complex Borrelia burgdorferi sensu lato (s.l.) transmitted by Ixodes ticks. At present, serology remains the main diagnostic tool for laboratory diagnosis of Lyme borreliosis. Recently, the PCR technique has been applied for diagnosis of B. burgdorferi s.l., but, until now, a reliable, easy-to-perform and sensitive method has not been described. Here we present a new PCR-based method for the detection of both B. burgdorferi s.l. and Borrelia genospecies DNAs in serum samples collected from patients showing Lyme disease symptoms. Of 265 serum samples of patients included in this study, 7.5% were positive, 1.9% was borderline and 90.6% were negative for antibodies against B. burgdorferi by enzyme-linked immunosorbent assay and Western blotting. The B. burgdorferi s.l. 16S rRNA gene was detected by PCR in all serum-positive and in two borderline samples. None of the serum-negative samples nor serum samples collected from healthy subjects gave positive PCR reactions. Of PCR-positive serum samples, 50% gave a positive reaction for Borrelia afzelii, 18% for Borrelia garinii and 23% for two Borrelia species. Two samples (9%) were not identified to species level. The new protocol could be considered to be reliable as neither false-positive nor false-negative reactions were recorded, and to be sensitive as it detects DNA from one bacterial cell.