The chromatographic behavior of human amniotic interferon on various affinity chromatography ligands was studied. Most of this interferon bound strongly to bovine plasma albumin-agarose, cibacron blue F3GA-agarose, concanavalin A-agarose and L-tryptophyl-L-tyrosine-omega-carboxyl-pentyl-agarose. After binding most of the interferon activity was eluted only with 50 percent ethylene glycol, showing the high hydrophobicity of this interferon. Smaller quantities could be recovered after phosphate-buffered saline elution or with increased salt concentration. On BPA-omega-carboxy-pentyl-agarose and omega-amino-hexyl-agarose, the majority of the biological activity was found in the break-through fraction (eluted with phosphate buffered saline) while some interferon was displaced with high salt or ethylene glycol. Increasing the salt concentration and lowering the pH was necessary to elute interferon from zinc chelate-agarose. These patterns indicate that human amniotic interferon is similar to human fibroblast (beta) interferon but different from human leukocyte (alpha) interferon. However, the heterogeneity displayed by amniotic interferon on bovine plasma albumin-agarose requires further investigation.