Zinc serves regulatory functions in cells and thus, several mechanisms exist for tight control of its homeostasis. One mechanism is storage in and retrieval from vesicles, so-called zincosomes, but the chemical speciation of zincosomal zinc has remained enigmatic. Here, we determine the intravesicular zinc-coordination in isolated zincosomes in comparison to intact RAW264.7 murine macrophage cells. In elemental maps of a cell monolayer, generated by microbeam X-ray fluorescence, zincosomes were identified as spots of high zinc accumulation. A fingerprint for the binding motif obtained by mu XANES (X-ray absorption near edge structure) matches the XANES from isolated vesicles; zinc is not free, but present as a complexed form (average coordination: 1.0 sulfur, 25 histidines 30 and 1.0 oxygen), resembling regulatory or catalytic zinc sites in proteins. Such Coordination enables reversible binding, acting as a 'zinc sink', facilitating the accumulation of high amounts of zinc against a concentration gradient. (C) 2009 Elsevier Inc. All rights reserved.