Humans are characterized by an unusual level of prosociality. Despite this, considerable indirect evidence suggests that biological kinship plays an important role in altruistic behaviour. All previous reports of the influence of kin selection on human altruism have, however, used correlational (rather than experimental) designs, or imposed only a hypothetical or negligible time cost on participants. Since these research designs fail either to control for confounding variables or to meet the criteria required as a test of Hamilton's rule for kin selection (that the altruist pays a true cost), they fail to establish unequivocally whether kin selection plays a role. We show that individuals from two different cultures behave in accordance with Hamilton's rule by acting more altruistically (imposing a higher physical cost upon themselves) towards more closely related individuals. Three possible sources of confound were ruled out: generational effects, sexual attraction and reciprocity. Performance on the task however did not exhibit a perfect linear relationship with relatedness, which might reflect either the intrusion of other variables (e.g. cultural differences in the way kinship is costed) or that our behavioural measure is insufficiently sensitive to fine-tuned differences in the way individuals view their social world. These findings provide the first unequivocal experimental evidence that kinship plays a role in moderating altruistic behaviour. Kinship thus represents a baseline against which individuals pitch other criteria (including reciprocity, prosociality, obligation and a moral sense) when deciding how to behave towards others.