The amino-acid sequence and three-dimensional structure of a calcium-binding protein prepared from carp muscle has been determined. This protein, designated carp-muscle calcium-binding protein B, is one of three closely related parvalbumins found in this tissue. The electron density map, calculated by heavyatom substitution crystallographic methods to 2.0-A resolution, reveals the orientation of most of the amino-acid side chains. The calcium coordination site consists of one glutamic- and three aspartic-acid carboxyl groups in a tetrahedral arrangement. The core of this spherical molecule is remarkably hydrophobic, with 8 of its 10 phenylalanine side chains packed in an approximate herringbone pattern. 52 of the 108 residues are in six alpha-helixes; there is no beta-pleated sheet. The acetylated amino-terminal alanine appears not to be accessible to solvent. All of the heavy-atom derivatives are bound at the sole cysteine. The properties of this protein suggest a relationship to troponin A of mammalian tissue.