Accurate random coil α‐proton chemical shift values are essential for precise protein structure analysis using chemical shift index (CSI) calculations. The current study determines the chemical shift effects of pH, urea, peptide length and neighboring amino acids on the α‐proton of Ala using model peptides of the general sequence GnXaaAYaaGn, where Xaa and Yaa are Leu, Val, Phe, Tyr, His, Trp or Pro, and n = 1–3. Changes in pH (2–6), urea (0–1M), and peptide length (n = 1–3) had no effect on Ala α‐proton chemical shifts. Denaturing concentrations of urea (8M) caused significant downfield shifts (0.10 ± 0.01 ppm) relative to an external DSS reference. Neighboring aliphatic residues (Leu, Val) had no effect, whereas aromatic amino acids (Phe, Tyr, His and Trp) and Pro caused significant shifts in the alanine α‐proton, with the extent of the shifts dependent on the nature and position of the amino acid. Smaller aromatic residues (Phe, Tyr, His) caused larger shift effects when present in the C‐terminal position (∼0.10 vs. 0.05 ppm N‐terminal), and the larger aromatic tryptophan caused greater effects in the N‐terminal position (0.15 ppm vs. 0.10 C‐terminal). Proline affected both significant upfield (0.06 ppm, N‐terminal) and downfield (0.25 ppm, C‐terminal) chemical shifts. These new Ala correction factors detail the magnitude and range of variation in environmental chemical shift effects, in addition to providing insight into the molecular level interactions that govern protein folding. © 2006 Wiley Periodicals, Inc. Biopolymers 85: 72–80, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com