Crystals of alkaline phosphatase (EC 3.1.3.1; M r 94,000) grown at pH 9.5 from 2.25 m-(NH 4) 2SO 4 with 5 × 10 −5 m-Zn and 10 −2 m-Mg present were analyzed by X-ray diffraction at pH 7.5 in 2.66 m-(NH 4) 2SO 4 with 10 −2 m-Zn and 10 −2 m-Mg present. The crystals are orthorhombic with a = 195.5 A ̊ , b = 168.3 A ̊ and c = 76.33 A ̊ , and the space group is I222. X-ray phases were determined by the multiple isomorphous replacement and anomalous dispersion method using K 2PtCl 4, KAu(CN) 2 and K 2OsO 4 derivatives. The electron density maps and analysis of metal binding sites reveal one molecule per asymmetric unit with an internal, non-crystallographic, 2-fold rotation axis relating the subunits. Each subunit contains a major α β domain with a seven-stranded β-sheet flanked by helices. The sheets are roughly coplanar but the general direction of the strands in each is at 20 ° to the rotation axis and thus 40 ° from each other. The helical content of the α β domain is approximately 27% of the 459 residues in the monomer and the β content is approximately 7%. The chains in a smaller domain are more convoluted and less easily characterized than in the α β domain. In both there is extensive monomer-monomer contact. Removal of the zinc and magnesium from the parent crystal produces a stable apoenzyme crystal and addition of cobalt at 10 −2 m or cadmium at 10 −2 or 5 × 10 −2 m reveals seven metal binding sites per dimer. The active centers are 32 Å apart and each is shown by anomalous dispersion data to contain two metal binding sites, A and B. The cadmium derivative refinement determined the A-B separation to be 4.9 Å. Comparison of the parent and apo structures by means of difference maps reveals the double metal site with Zn at A and probably Mg at B. A prominent, partially resolved peak centered 7 Å away is interpreted as a stabilization of the backbone in this position by the metal ion co-ordination to a side-chain. Several negative peaks within 10 Å of the metals indicate local differences between apo and native structures but no significant differences are seen in the other parts of the molecule. At 5 × 10 −2 m-Cd two metal sites (D and D′) are found 25.5 Å from the active center, on the surface of the minor domain. They are related to each other by the molecular 2-fold axis with a D-D′ distance of 25 Å. The seventh Cd site, E, is 20 Å from the active center, on the major domain, near a crystalline contact region, and devoid of any molecular symmetry mate. The apparent dissociation constants for cadmium at the A, B and D sites (and A′, B′, D′) are 3 × 10 −3 m, 1.5 × 10 −1 m and 1.3 × 10 −2 m, respectively. Thus in these conditions cadmium is seen to distribute between A and B sites when the combined stoichiometry is two metal ions per dimer.