Mycobacterium avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease, mainly is transmitted to susceptible calves via the oral-fecal route. Map also can be transmitted transplacentally, thus detection of Map in such infected fetuses would allow for more efficient culling of all infected animals in a herd, the preferred method of disease control. In this study, a percutaneous technique for the sterile collection of allantoic fluid during late gestational pregnancy on the locally sedated standing animal was employed for the analysis of Map DNA via nested PCR (nPCR). A total of 12 infected pregnant Holstein cows with signs of clinical Johne's disease were studied using IS900 and primers P90,P91 and J1,J2 for the nPCR. Antemortem samples studied were blood and allantoic fluid from the dam and after necropsy intestinal and mesenteric lymph node tissues from the dam, placental fluid, and fetal tissues such as liver, spleen, brain, and cotelydon. Nested PCR performed on the allantoic fluid collected multiple times antemortem from three cows greater than 7 months into pregnancy were negative each time. Allantoic fluid collected at necropsy was positive on nPCR for Map DNA in 2 additional cows at mid-gestion.The spleens and liver of 5 fetuses (including one set of twins) and the lung, liver and brain of another fetus amplyfied on nPCR. One of the 12 cows had microscopically demonstrable Map bacilli in the placentome were verified by nPCR to be Map DNA. A total of 6 fetuses (50%) were PCR positive at least on one tissue. These results demonstrate that Map can be transmitted in-utero, even though bacterial DNA may not appear in the allantoic fluid very often. This observation may have implications to consider infected dams and their offspring for the test and cull program in the effort to control Johne' disease.