As an undergraduate at Oregon Agricultural College (now Oregon State University), Pauling studied chemical engineering. When he arrived at California Institute of Technology (Caltech) for graduate school, Arthur A. Noyes, head of the Research Laboratory of Physical Chemistry, suggested to Pauling that he learn x-ray crystallography. X-ray crystallography allows an investigator to see the three-dimensional shape of the molecule analyzed. Between 1923 and 1925, while a graduate student at Caltech, Pauling published seven papers on crystal structures, five of which he included in his thesis to obtain his Ph.D. in Chemistry. In the years ahead, Pauling would use x-ray crystallography to determine the atomic structure of organic compounds, especially proteins.

In 1932 Pauling began analyzing not only inorganic, but also organic molecules. How did Pauling gain an interest in organic substances after training and working with inorganic compounds for over ten years? There are multiple possibilities as to why Pauling's interests shifted. Funding might have motivated Pauling to some extent. In 1932 he applied for a grant from the Rockefeller Foundation with the purpose of primarily examining the structure of inorganic molecules. However, in his grant proposal, Pauling mentioned that his inorganic researches might aid knowledge on organic substances and specifically named "proteins, haemoglobin and other complicated organic substances." Pauling's comment attracted the attention of the Rockefeller Foundation's Warren Weaver, who encouraged scientists, such as Pauling, who integrated scientific disciplines. The Rockefeller Foundation gave Caltech enormous sums of money during the twentieth century and helped Caltech to become a leading scientific institution in the United States.

Other reasons, besides funding, motivated Pauling's new path. Pauling said he took the next logical step by moving from the less complex inorganic molecules to more complex organic compounds. Arthur A. Noyes directed the department with a focus upon biological matters by striving to bring chemistry and biology together. Caltech's small size allowed the different departments to share information and cultivate cross-disciplinary interests. In this vein, Caltech added their biology department in 1929 under the direction of geneticist Thomas Hunt Morgan. Pauling not only attended a weekly lecture on genetics given by Morgan, but also discussed research projects with Morgan and his colleagues.

By early 1935 Pauling enthusiastically pursued hemoglobin research.