Validation is the key to the regulatory status of alternative methods. A series of questions are put, to which answers are given, including the following: What is validation? What is meant by "relevance", "reliability" and "purpose"? Why and when is formal validation necessary? What comes before and after a formal validation study? How have validation criteria been defined, and to what extent have they been harmonized internationally? How are validation studies set up, managed and funded? What is a test? Do prediction models have to be validated? What is prevalidation? What is acceptance, and who is responsible for acceptance? How are validation studies reported? How should a validated test be defined and recognized? Must all new tests be validated? Are the same standards being applied to new in vitro and new in vivo tests? Has validation been successful so far? What can be done to improve validation? Is validation helping or hindering the development of in vitro toxicology and the implementation of the 'Three Rs' of Russell & Burch?