In the Netherlands an eradication program of Mycobacterium paratuberculosis (Mptb) is considered. The design of this program depends highly on the herd and cow prevalence of Mptb. To estimate this prevalence, out of the 28918 dairy herds with > 20 cattle older than 2 years, a sample of 460 herds was selected. Herds were randomly selected in the four regions (North, East, West, and South) with an equal proportion. In total 378 farmers, 91-97 per region, decided to participate. Serum samples of all cows older than 3 years of age were collected in the period July - October 1998. The farmers was asked if the cattle was vaccinated against paratuberculosis. Serum samples were tested using the Ubitech (Idexx) absorbed ELISA. Serum samples with a Sample to Positive ratio > 0.3 were regarded as positive. Out of a total of 15822 cows, 520 animals were tested positive. Taking into account the regional distribution of the number of herds as well as the herd size, it was calculated that 3.3% of the cattle in the Netherlands was serologically positive. The percentage positive cows differed between regions. In the Northern region, for example, 5.0% of the cows were tested as positive, whereas in het Western region only 2.2% were tested positive. The percentage herds with at least one serologically positive cow was calculated to be 55%.In the Northern region the percentage herds with at least one positive cow was the highest (69%) and in the Western region the lowest (44%). Of the serologically positive herds (n = 206) 96 herds had only one positive cow whereas 49 herds had 2 positive cows. Eleven of the herds tested, were vaccinated against paratuberculosis and had a total of 95 positive cows (range 1-28 animals per herd). Eight of these herds belonged to the Northern region.