Cancerous transformation entails major biochemical changes including modifications of the energy metabolism of the cell, e.g. utilisation of glucose and other substrates, protein synthesis, and expression of receptors and antigens. Tumour growth also leads to heterogeneity in blood flow owing to focal necrosis, angiogenesis and metabolic demands, as well as disruption of transport mechanisms of substrates across cell membranes and other physiological boundaries such as the blood-brain barrier. All these biochemical, histological and anatomical changes can be assessed with emission tomography, X-ray computed tomography (CT), magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). Whereas anatomical imaging is aimed at the diagnosis of brain tumours, biochemical imaging is better suited for tissue characterisation. The identification of a tumoural mass and the assessment of its size and vascularisation are best achieved with X-ray CT and MRI, while biochemical imaging can provide additional information that is crucial for tumour classification, differential diagnosis and follow-up. As the assessment of variables such as water content, appearance of cystic lesions and location of the tumour are largely irrelevant for tissue characterisation, a number of probes have been employed for the assessment of the biochemical features of tumours. Since biochemical changes may be related to the growth rate of cancer cells, they can be thought of as markers of tumour cell proliferation. Biochemical imaging with radionuclides of processes that occur at a cellular level provides information that complements findings obtained by anatomical imaging aimed at depicting structural, vascular and histological changes. This review focusses on the clinical application of anatomical brain imaging and biochemical assessment with positron emission tomography, single-photon emission tomography and MRS in the diagnosis of primary brain tumours, as well as in follow-up.