Hemoglobin fascinated Pauling throughout his life. He began experimenting with it in the early 1930s and continued to write about it until his death in 1994. Since Pauling found hemoglobin intriguing, he learned all he could about it, chemically, biologically and structurally. The exact structure of hemoglobin was determined in 1959 by Max Perutz of Cambridge University; however, prior to then Pauling knew as much as anyone about its structure.

In 1935, Pauling chose hemoglobin as one of his first organic substances to investigate. Several motives elucidate his decision. Hemoglobin is easily obtainable. During this time, the 1930s and 1940s, no one knew for sure which substance in the human body controlled heredity, but most scientists believed proteins, such as hemoglobin, held the secret to life. Proteins are fragile substances to study, and hemoglobin's accessibility enhanced its allure. Hemoglobin can be studied by x-ray crystallography, a technique Pauling had learned while working with inorganic compounds as a graduate student. Also, hemoglobin is a rather large macromolecule, but it can be broken down and analyzed in sections – which is exactly what Pauling did.