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DEPFET detectors are silicon active pixel sensors for X-ray imaging spectroscopy. They will be used for the MIXS instrument of BepiColombo planetary mission and they are foreseen for the Wide Field Imager of the International X-ray Observatory currently in study with ESA, NASA and JAXA. New DEPFET matrixes with 256 × 256 pixels of 75 μm pitch have been produced, mounted on ceramic boards with dedicated front-end electronics and integrated in a new set-up able to acquire large-format images and spectra. Excellent homogeneity has been observed on two samples. Energy resolution as low as 129 eV FWHM at 5.9 keV has been obtained including all single events of the matrix back illuminated at -40°C and read out at a 300 frames per second rate. Experimental methods and results are reported.