A brief description is given of the events surrounding the development of biological X-ray microanalysis during the last 30 years, with particular emphasis on the contribution made by research workers in Cambridge, UK. There then follows a broad review of some applications of biological X-ray microanalysis. A more detailed consideration is given to the main thrust of current procedures and applications that are, for convenience, considered as four different kinds of samples. Thin frozen dried sections which are analyzed at ambient temperatures in a transmission electron microscope (TEM); semithin frozen dried sections which are analyzed at low temperature in a scanning transmission electron microscope (STEM); thick frozen hydrated sections which are analyzed at low temperature in a scanning electron microscope (SEM), and bulk samples which are analyzed at low temperature in the same type of instrument. A brief outline is given of the advantages and disadvantages of performing low-voltage, low-temperature X-ray microanalysis on frozen hydrated bulk biological material. The article concludes with a consideration of alternative approaches to in situ analysis using either high-energy beams or visible and near-visible photons.