In the Netherlands, vaccination against paratuberculosis in cattle is performed on a limited scale. Because of its interference with the diagnostics for bovine tuberculosis, vaccination is restricted to herds with a high prevalence of paratuberculosis and is meant to aid in the economical survival of the farm. At present plans are being made to begin with a nationwide certification program for paratuberculosis. Since serological methods are likely to part of the first steps in any conceivable certification program, herds that have been vaccinated in the past are likely to encounter problems when entering such a program. The aim of this study was to evaluate the immune response resulting from vaccination using a heat killed vaccine. Two farms, one with 102 animals and one with 208 animals were selected. On both the vaccination program started more than 9 years ago; the larger farm is still vaccinating, whereas the smaller one stopped vaccinating two years ago. The humoral immune response was evaluated in all animals using the CFT test and the absorbed ELISA, the T-cell response using the gamma-interferon assay with bovine and avian PPD and Johnin as stimulants. Furthermore, faecal culture was performed on all animals to assess the influence of possible lingering paratuberculosis infection in the herds on the immune status of the animals. The data obtained show a prolonged effect of the vaccination on both the cellular- and the humoral immune response, in particular to the avian- and paratuberculosis antigens. Implications of these results for the certification of these herds will be discussed.