Cross-priming is an important mechanism to activate cytotoxic T lymphocytes (CTLs) for immune defence against viruses and tumours. Although it was discovered more than 25 years ago, we have only recently gained insight into the underlying cellular and molecular mechanisms, and we are just beginning to understand its physiological importance in health and disease. Here we summarize current concepts on the cross-talk between the immune cells involved in CTL cross-priming and on its role in antimicrobial and antitumour defence, as well as in immune-mediated diseases.