Elemental microanalysis of biological material in the frozen-hydrated state using in-vacuum proton induced X-ray emission is described for the first time. For this purpose, a commercially available cryotransfer system was modified and coupled to the experimental chamber of the nuclear microprobe (NMP). The analyzed material was frozen in propane cooled by liquid nitrogen, fractured, carbon coated, and transferred onto the cold stage (100 K) of the nuclear microprobe chamber. Micro-PIXE and simultaneous proton backscattering was performed using a 3 MeV proton beam. Quantitative results were obtained by the standardless method, and tested using 20% gelatin standards. Monitoring of the gas composition inside the system by means of mass spectrometry performed before, during, and after proton bombardment showed good stability of the analyzed material for proton currents not exceeding 150 pA. Average concentrations of light elements (C, N, O, and indirectly H) were also obtained by the proton backscattering technique. No losses of elements measurable by particle-induced X-ray emission (PIXE) during proton irradiation were found during repetitive, short analyses of the same micro areas of gelatin standards. Measurements of thick sections of selected plant and animal material in the frozen-hydrated state-leaf sections of the plant Senecio anomalochrous Hilliard (Asteraceae) and larvae of Chysolina pardalina Fabricius (Chrysomelidae)-showed very good preservation of morphology and elemental distribution. Limits of detection of the order of a few micro g g(-1) were obtained for most elements.