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An irradiation facility, able to expose cellular and subcellular targets to a precise number of particles, has been developed at CENBG for applications in radiobiology. The development of this facility was based on an existing horizontal focused microbeam developed in the early 90’s for material analysis. The focusing properties of the line allow the delivering of proton or alpha particle beams in the 1–3.5 MeV energy range with a spatial resolution down to about 1 μm under vacuum. For irradiation of living cells, a removable stage has been developed to extract the beam into air while preserving the analytical capabilities of the microbeam line under vacuum. This stage includes a high resolution epifluorescence microscope for online visualization of the cells and a motorized stage for cell positioning. Single particle control is ensured by a fast electrostatic deflector triggered by the signal induced by the particles through a transmission detector just before reaching the target. A dedicated software, based on an object-oriented architecture, has been designed to control the entire experiment. This includes semiautomatic calibration procedures (necessary to achieve the micron precision) and semiautomatic irradiation procedures used for targeting a large number of individual cells. In air irradiation of solid track detectors has permitted us to estimate that 99.5% of the particles are delivered on the target at a distance lower than 5 μm from the beam center when an alpha particles beam is used. The targeting precision of the overall irradiation procedure, which reflects the alignment precision of the beam center with the target center, has been estimated to be within ±2 μm. First experiments involving cells in culture have permitted to estimate an irradiation rate of 2000 cells per hour. This article presents the overall experimental facility and the tests performed for its validation for the irradiation of individual cells in their culture medium. [ABSTRACT FROM AUTHOR]