Two forms of calbindin-D9k have sometimes been observed within a single tissue. Sequencing of these proteins has been complicated by the presence of blocked amino termini. Tandem mass spectrometry is a powerful tool for comparing related proteins, and its use does not depend upon an unblocked amino terminus. In the present studies, calbindin-D9k was purified from the intestines of mice (270 animals per purification) by use of gel permeation chromatography and two preparative electrophoresis steps in the presence and absence of EDTA. The purified protein appeared to be homogeneous following electrophoresis under nondenaturing conditions, but two components were identified by sodium dodecyl sulfate-gel electrophoresis and immunoblotting. Two forms of the protein were isolated by reverse-phase high performance liquid chromatography. In each of three preparations, the average ratio of the major:minor isoforms was 2:1. The major form contained 77 amino acids and lacked the amino-terminal serine found in 78-amino acid calbindins from rat and pig. The amino acid sequence was identical with the deduced sequence reported for rat intestinal calbindin-D9k in 73 of 77 positions. In the minor form, a glutamine was found in a location between Lys-43 and Ala-44 of the major form and between the two calcium binding sites of the protein. The minor form was otherwise identical with the major form, including the presence of a blocked amino terminus. The inserted glutamine was located at the site of an intron in the rat calbindin gene, suggesting the possibility that alternative splicing produced the two forms of calbindin-D9k. The functional significance of an inserted amino acid between the two calcium binding sites remains to be explored.