We have explored immobilized metal affinity chromatography as a means of resolving alpha-fetoprotein from its homologous albumin, a problem perennially encountered in the purification of an alpha-fetoprotein or its detection. Human alpha-fetoprotein and human serum albumin were chromatographed on immobilized iminodiacetic acid charged with either Co2+, Ni2+, Cu2+, or Zn2+. Neither human alpha-fetoprotein nor human serum albumin displayed any affinity for Co2+ and Zn2+. However, both proteins were bound to Cu2+ and were partially resolved by affinity elution with imidazole. By contrast, human alpha-fetoprotein and human serum albumin were completely resolved on immobilized Ni2+. Similar results were obtained using bovine alpha-fetoprotein and bovine serum albumin. The resolution of an alpha-fetoprotein from serum albumin should aid the purification of alpha-fetoprotein from a biological fluid containing overwhelming quantities of albumin, for example, serum. Importantly, the separation of human alpha-fetoprotein from human serum albumin may improve and help maintain the accuracy of immunoassays for alpha-fetoprotein, making the chromatography on immobilized Ni2+ a valuable diagnostic tool.