In 114 Ixodes ricinus ticks from 1 district of Lublin region (eastern Poland) examined by dark field microscopy method, the presence of motile spirochetes, morphologically corresponding to Borrelia species was detected in 8.8% of the total examined ticks. The highest infection rate was noted among females (16.7%), much lower in males (7.1%) and nymphs (4.0%). Examination of 550 ticks collected from 3 districts of the Lublin region by polymerase chain reaction (PCR) showed that 5.3% of the total number of ticks were infected with Borrelia spirochetes. The highest rates of Borrelia infection were observed in Ixodes ricinus ticks from the Zamość and Lublin districts (9.6% and 4.7% respectively). In the Włodawa district, only 2.4% ticks showed the presence of Borrelia DNA. In contrast to the results obtained by dark field microscopy method, the highest infection rate was noted in males (11.2%), followed by females (6.9%) and nymphs (1.7%). 57 Ixodes ricinus ticks collected from 3 Lublin districts (Lubartów, Lublin, Radzyń Podlaski) were tested for the presence of tick-borne encephalitis virus (TBEV) by the inoculation of 5-week-old Swiss mice, followed by blind passages and inoculation of GMK cell culture with brain suspension of the infected mice. One strain of TBEV was isolated from a pool of 24 I. ricinus ticks collected from the Radzyń Podlaski district. The minimum infection rate of ticks from this district with TBEV was estimated as 4.2%, and in the total area of Lublin region estimated as 1.8%.