Expression of interferon (IFN) during embryogenesis of the Syrian hamster has been characterized with respect to (1) the antiviral activity to IFN; (2) the activity of the IFN-induced enzyme, 2',5'-oligo A synthetase; (3) the subpopulation of IFN producing cells, and (4) the molecular structure of the elaborated IFN. These components of the IFN system were examined in cell cultures derived from embryos excised at 8-13 days of gestation and determined as a function of both in utero gestation and in vitro passaging. The antiviral responsiveness of nine-day gestation cultures (9 dgc) was 1/4-1/6 of that of 13 dgc, but in vitro passaging increased the responsiveness as did in vivo development. IFN enhancement of the synthetase level in 9 dgc was only minimal when compared with that in 13 dgc. However, the 9 dgc contained an unusually high basal level of the enzyme. During in vivo development and in vitro passaging, the basal levels of the enzymes progressively declined while the IFN-induced levels progressively increased. IFN production in embryo cells following induction by Newcastle disease virus differs substantially, depending on the gestational age of the cells. Using an agarose-overlay "zone of protection" assay, 8 dgc were found to contain 10-12 times the number of cells producing zones of protection than 13 dgc. Passaging of 9 dgc cells reduced the number of zones to the level of the 13 dgc, but had no effect on 13 dgc. Chromatographic analysis of IFN produced by 9 dgc and 13 dgc revealed the presence of an additional, unique species of "embryonic" IFN in 9 dgc which was not observed in IFN from 13 dgc. These observations suggest that the expression of various components of the IFN system are under developmental control during embryogenesis.