Antiviral factors present in cultures of rabbit peritoneal macrophages or rabbit kidney (RK) cells infected with Newcastle disease virus (NDV) and those in cultures of uninfected macrophages all fulfilled the biological and physicochemical criteria for classification as interferons. Virus-induced macrophage and RK interferons were slightly more stable to heat or acid than "spontaneously produced" or endotoxin-induced macrophage interferon. Interferon activity in serum of NDV-infected rabbits was decidedly more labile than NDV-induced macrophage interferon. However, these differences in lability were too slight to serve as a useful basis for distinguishing one rabbit interferon from another. Rabbit interferons from various sources could be differentiated by filtration through Sephadex G-100 and their molecular weights estimated by comparison with elution profiles of a series of marker proteins of known molecular weight. Each of four different preparations of rabbit interferons was found to contain more than one molecular component. Elution peaks for three NDV-induced interferons were equivalent to the following molecular weights: RK approximately 44,000-45,000 and > 134,000 (variable and < 1% when present); macrophage approximately 37,000, 44,000-45,000, and > 134,000 (variable and <1% when present); and serum approximately 50,000-52,000 and > 134,000 ( approximately 10% and heat labile). NDV-induced serum interferon may also contain another molecular component of mol wt approximately 45,000 represented by a trailing shoulder from the major 51,000 mol wt peak. Endotoxin-induced macrophage interferon proved to be polydisperse. Sephadex filtration of this interferon did not reveal clear and consistent elution patterns, partially owing to its low initial titer and lability. However, variable peaks of biological activity could be detected in Sephadex fractions equivalent to approximate molecular weight values of > 134,000, 72,000-78,000, 33,000-38,000, 28,000-30,000, and possibly a component of 42,000-45,000. A major component of mol wt approximately 37,000 was present in all samples of endotoxin-induced macrophage interferon. The other constituents may be biologically active subunits or polymers. These data indicate that rabbit macrophages produce two primary kinds of interferon: (a) an RK-like component of mol wt approximately 45,000 that is synthesized in greatest amount after viral induction, and (b) a different species of mol wt approximately 37,000 that can also be synthesized in the absence of viral induction. The presence of major interferon constituents of mol wt approximately 51,000 and > 134,000 in rabbit serum after viral induction suggests that macrophages are not the principal interferon-producing cells that respond to intravenous injection of NDV.