Microdeletions of the Y chromosome are the second most frequent genetic cause of spermatogenetic failure in infertile men after the Klinefelter syndrome. The molecular diagnosis of Y-chromosomal microdeletions is routinely performed in the workup of male infertility in men with azoospermia or severe oligozoospermia. Since 1999, the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN) support the improvement of the quality of the diagnostic assays by publication of the laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions and by offering external quality assessment trials. The present revision of the 1999 laboratory guidelines summarizes the results of a ‘Best Practice Meeting’ held in Florence (Italy) in October 2003. The basic protocol for microdeletion screening suggested in the 1999 guidelines proved to be very accurate, sensitive and robust. In the light of the recent advance in the knowledge of the Y chromosome sequence and of the mechanism of microdeletion it was agreed that the basic 1999 protocol, based on two multiplex polymerase chain reactions each covering the three AZF regions, is still fully valid and appropriate for accurate diagnosis.