We describe a simple technique for non-invasively determining the absolute concentration of deoxyhaemoglobin (Hb) in living tissue using near infrared spectroscopy (NIRS). The technique uses second-differential spectroscopy to determine the relative concentration of Hb to tissue water by fitting of the spectral features of these two chromophores in the 710 nm to 840 nm region of the near infrared (NIR) spectrum. Since the concentration of tissue water is generally known with accuracies of a few per cent, one can then obtain the absolute concentration of Hb ([Hb]). The validity and likely accuracy of the technique is assessed by applying it to artificially generated NIRS data. Some clinical validation is presented by comparing, during an in vivo study, changes in [Hb] obtained by this method and those calculated using more conventional techniques of relative quantification. Finally, we discuss the likely clinical significance of the measurement of absolute Hb concentration.