Chicken intestinal calbindin‐D28k is an intracellular protein which is believed to have a fundamental role in vitamin D‐mediated transport of calcium. A mapping approach based on 252Cf plasma desorption mass spectrometry (PD mapping) was used to screen the DNA‐deduced sequence of calbindin‐D28k for sequence changes and posttranslational modifications. In the PD mapping experiment, purified calbindin‐D28k was cleaved with cyanogen bromide and the resulting peptides were subjected to PD mass spectrometric analysis either as a mixture or as high‐performance liquid chromatography isolated fractions. The DNA‐derived primary structure of calbindin‐D28k was confirmed by rapid PD mass spectral identification of the CNBr peptide fragments, and the nature of the N‐terminal blocking group was readily determined to be an acetyl group. The relatively non‐destructive nature of the PD mass spectrometric analysis allowed the mapping of the N‐terminal peptide through an additional in situ V8 protease enzymatic reaction.