The structural investigation of noncrystalline, soft biological matter using x-rays is of rapidly increasing interest.Large-scale x-ray sources, such as synchrotrons and x-ray free electron lasers, are becoming ever brighter and make the studyof such weakly scattering materials more feasible. Variants of coherent diffractive imaging (CDI) are particularly attractive, as theabsence of an objective lens between sample and detector ensures that no x-ray photons scattered by a sample are lost in alimited-efficiency imaging system. Furthermore, the reconstructed complex image contains quantitative density information,most directly accessible through its phase, which is proportional to the projected electron density of the sample. If applied inthree dimensions, CDI can thus recover the sample’s electron density distribution. As the extension to three dimensions isaccompanied by a considerable dose applied to the sample, cryogenic cooling is necessary to optimize the structural preservationof a unique sample in the beam. This, however, imposes considerable technical challenges on the experimental realization.Here, we show a route toward the solution of these challenges using ptychographic CDI (PCDI), a scanning variant of coherentimaging. We present an experimental demonstration of the combination of three-dimensional structure determination throughPCDI with a cryogenically cooled biological sample—a budding yeast cell (Saccharomyces cerevisiae)—using hard (7.9 keV)synchrotron x-rays. This proof-of-principle demonstration in particular illustrates the potential of PCDI for highly sensitive, quantitativethree-dimensional density determination of cryogenically cooled, hydrated, and unstained biological matter and pavesthe way to future studies of unique, nonreproducible biological cells at higher resolution.