The 13C chemical shifts for all of the protonated carbons of the 20 common amino acid residues in the protected linear pentapeptide Gly-Gly-X-Gly-Gly have been obtained in water at low pH as well as in aqueous solution containing 10, 20 and 30% acetonitrile or trifluoroethanol. Dioxane was used as an internal reference and its carbon chemical shift value was found to be 66.6 ppm relative to external TMS in water. Comparison of the different referencing methods for 13C chemical shifts in organic cosolvent mixtures showed that an external standard (either TMS or TSP capillary) was the most appropriate. In the present study, external TSP was chosen to define the 0 ppm of the 13C chemical shift scale. When the difference in referencing the dioxane carbon resonance is taken into account, the carbon chemical shift values of the amino acids in aqueous solution are similar to those previously reported (Richarz and Wüthrich (1978) Biopolymers, 17, 2133-2141; Howarth and Lilley (1979) Prog. NMR Spectrose., 12, 1-40). The pentapeptides studied were assumed to be in a random coil conformation and the measured 13C chemical shifts were used as reference values to correlate carbon chemical shifts with the secondary structure of two well-characterized peptides, bombesin and the 1-29 amino acid fragment of Nle27 human growth hormone-releasing factor. In both cases, the C alpha chemical shifts exhibited a characteristic positive deviation from the random coil values, which indicates the presence of alpha-helices.