Identification of chemical species at a subcellular level is a key to understand the mechanisms involved in the biology of chemical elements. When performed with a microbeam, X-ray absorption near-edge structure (micro-XANES) enables the direct speciation analysis of oxidation states in subcellular compartments avoiding cell fractionation and other preparation steps that might modify the chemical species. Here we report the principal characteristics in terms of spatial resolution, detection limit, reproducibility, and repeatability of a micro-XANES experimental setup based on Kirkpatrick-Baez X-ray focusing optics that maintains high flux of incoming radiation (>10(11) photons/s) at micrometric spatial resolution (1.5 x 4.0 microm2). Applications and limitations of this setup are illustrated by examples of iron and arsenic absorption spectra obtained from the cytosol, nucleus, and mitochondrial network of cultured cells. A better repeatability and sensitivity with no oxidation state modification and minimal beam damage is achieved when cells are analyzed in a frozen hydrated state, as compared to freeze-dried cells. This original experimental setup can now be applied for the direct speciation analysis of most trace elements at the subcellular level.