Cytotoxic nucleoside analogues and nucleobases were among the first chemotherapeutic agents to be introduced for the medical treatment of cancer. This family of compounds has grown to include a variety of purine and pyrimidine nucleoside derivatives with activity in both solid tumours and malignant disorders of the blood. These agents behave as antimetabolites, compete with physiological nucleosides, and interact with a large number of intracellular targets to induce cytotoxicity. Progress has recently been made in the identification and characterisation of nucleoside transporters and the enzymes of nucleoside metabolism. In addition, there is now greater understanding of the molecular mechanisms of anticancer nucleoside activity, which provides opportunities for potentiating their antitumour effects. Strategies to optimise intracellular analogue accumulation and to enhance cancer-cell selectivity are proving beneficial in clinical trials.