RFLP analysis has been used for the study of epidemiology of paratuberculosis. In the present study, DNA from 336 M. paratuberculosis strains were examined by RFLP using restriction endonuclease PstI (in selected 306 strains restriction endonuclease BstEII was used in parallel) and a specific probe IS900. After digestion with restriction endonuclease PstI, the strains were differentiated into 9 different RFLP types named A (n=90), B (n=164), C (n=4), D (n=16), E (n=53), F (n=1), g (n=1), H (n=6) and I (n=1). After digestion with the restriction endonuclease BstEII, the strains were differentiated into 14 RFLP types named according to previously presented results by Collins et al (J. Clin Microbiol. 28:1591-1596): C1 (n=173), C2 (n=2), C3 (n=8), C5 (n=1), S1 (n=4) and I1 (n=2). Other strains of 8 RFLP types were provisionally named C8-C15 (n=116). Systematic long term investigation allowed us to perform a regressive epidemiologic study on spreading of the infection in cattle herds of the Czech Republic during the recent 9 years. In 5 farms located in one region, the isolated strains were of the same RFLP type during 3 years. High regional homogeneity can be explained by the same infection source in pastured cattle. Since 1988 RFLP types A-E, I, and C1-2, C8-10, C12-13 and S1 of M. paratuberculosis have been isolated from ruminants in the Czech Republic. The variety of isolated RFLP types indicates different infection sources in ruminants kept in the Czech Republic. Identification of different RFLP types allowed us to the monitor both vertical and horizontal spreading of the infection in herds with imported cattle infected with strains of different RFLP types and in infection-free cattle bred in the Czech Republic. It was found, using the same methods, that calves up to the age of 1-2 months after birth being in contact with infected cows are the most susceptible.