The localization of in ovo injected substances in the chicken was investigated. We determined that localization is dependent on the nature of substances and the time of in ovo injection. In ovo injections with soluble bromodeoxyuridin (BrdU), particulate colloidal carbon, 40 nm fluorescent microspheres, and live Infectious Bursal Disease Virus (IBDV) were performed with a 25-mm (1-in) needle at Days 16 and 18 of incubation (DI-16 and DI-18, respectively). Localization of injected substances was determined in several organs using immunocytochemical methods. At DI-16, approximately 50% of the substances were detected in the organs; therefore, the localization of substances was not consistent. At DI-18, the substances were injected into the amnion. The substances entered the embryo by the mouth and were ingested into the intestinal and respiratory tract. All substances reached the lungs of the embryo via the trachea and the bronchi and were absorbed by the gas exchange tissue. In addition, the substances were absorbed by the bursa. Particulate colloidal carbon and microspheres remained in the organs where they were taken up initially for the rest of time of the experiment. Live IBDV, however, was distributed to other organs of the embryo. Soluble BrdU was found in all investigated organs of the embryo in high amounts. These results demonstrate that in ovo injection at DI-18 is an effective route to introduce substances into the chicken embryo, whereby the characteristics of the substance determine its final localization.