DEPFET detectors are silicon (Si) active pixel sensors designed and manufactured in the Max-Planck-Institut semiconductor lab. Their high spatial resolution and high energy resolution in X-rays make them attractive for particle tracking in colliders and for X-ray astronomy. This technology is 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 setup able to acquire large-format images and spectra. Excellent homogeneity has been observed. Energy resolution as low as 127 eV FWHM at 5.9 keV has been obtained including all single events of the matrix back illuminated at -45<;°C and read out at a 300-frames/s rate. This paper presents experimental methods and results.